Team:Goettingen/NoteBook w2

From 2013.igem.org

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Revision as of 05:21, 20 August 2013

14th

Observation of Back-up plates and preparation of media

Observation of Back-up plates

- Part 1, C1 and C2 probably very light pink

- Part 2, C2 and C3 became light pink, but C1 stayed white (compare with restriction analysis 13.6.13)

Preparation of media for inoculation on Sunday (by Katrin Gunka)

- For Plate Reader assay: 4 ml LB + 4 μl Cm or Amp for C1, C2 and C3 of parts 1 – 4 and part 6, negative control DH5α in 4 ml LB

- For Plasmid Mini-Preparation: 10 ml LB + 10 μl Amp for inoculation of C2 Part 2

Fold ↑

13th

Test restriction of plasmids containing parts 1 – 8

Test restriction of plasmids containing parts 1 – 8

- Reactions/Master Mixes: see Excel-sheet “dropbox > iGEM > Reporter-Team > digestion system 2hour”

- Plasmids: parts 1 – 6 (plasmids purified on 7.6.13) and part 7, 8 (plasmids purified on 11.6.13)

- 1x reaction with single enzyme:

Component	Volume
Enzyme	0.5 µl
Buffer 10x	1 µl
Plasmid DNA	200 ng
Total volume	10 µl

è For double digestion:

0.5 µl of each enzyme in case of EcoRI and PstI;

for SpeI and PstI, ratio 1:2 is recommended à 0.5 µl SpeI and 1 µl PstI used

- Incubation at 37°C for1.5 hours

- Additon of 5xDNA-Loading Dye to whole reaction

- Loading of 8 µl on agarose gel (~1.5 %, 1xTAE), QuickLoad 1 kb ladder as marker

- Run at 200 V for 1.5 h in 1xTAE

- EtBr staining ca. 45 min, destaining in water ca. 30 min

- UV detection

Expected Fragments:

Part no.	Fragments for PstI and SpeI (bp)	Plasmid size (linearization with PstI or SpeI)(bp)
1	~900 + 2000	2948
2
3
4
Part no.	Fragments for PstI and EcoRI(bp)	Plasmid size (linearization with PstI or EcoRI)(bp)
5	??? (if RFP gene à same pattern as expected for parts 1 – 4; if no RPF gene à same pattern as expected for part 8)
6	917 2029	2946
7	140 2050	2199
8	53 2038	2091

Gel: See ppt-file “dropbox > iGEM > Reporter-Team > Geldoc“

Conclusions:

è Partial digestion (next time: longer incubation time, less plasmid…)

è For parts 1, 3, 4, 6, 7 and 8, the expected bands were observed (ok)

è Band pattern of part 5 resembles that expected for parts 1 – 4 à this plasmid contains RFP gene

è In case of part 2, SpeI was unable to cut the plasmid à no cleavage for SpeI single digest + linearization for SpeI/PstI double digest à plasmid contains probably something else (SpeI/XbaI scar at actual SpeI site?)

è For cloning: terminator (part 7) and strong RBS (part 8) might be difficult to extract from the gel, since they are very short à think of other cloning strategy without gel extraction (e.g. “play” with resistances of plasmid backbones to digest backbone after cutting out part…)

Preparation of DarR PCR samples for sequencing

- 4 µl of DarR seq PCR product of reaction 1 + 1 µl of iGEM_34 (fwd) or iGEM_35 (rev)

Tubes:

kgun_1 à iGEM_34

kgun_2 à iGEM_35

- Sequencing at G2L

Fold ↑

11th

Miniprep of Part7,8 and DarR sequencing PCR clean-up

Cryo-Stocks of E. coli transfomants (8, 7 C1)

Stored in -70 °C (red box)

Plasmid Mini-Preparation of parts 8, 7 C1

- harvesting of ca. 5 ml pellets in 2 ml Epis for future Plasmid Mini-Preparation à stored at – 20 °C in red box

- harvesting of remaining culture for today’s prep

è Performed as on 7.6.13

NanoDrop – Plasmid concentrations

Part no.

c(DNA)

[ng/µl]

A₂₆₀/A₂₈₀

A₂₆₀/A₂₃₀

7

67.0

1.98

2.25

8

62.7

1.89

2.08

DarR seq PCR purification

- for DarR seq PCR reactions 1 - 4

- with QIAquick PCR purification Kit (Qiagen), according to quick start manual

- ca. 50 µl of PCR reaction + 250 µl of PB buffer

reactions 2 and 4 turned violet à addition of 10 µl NaAc 3.3 M, pH = 5.0

- elution: 30 µl pre-warmed HPLC water directly applied to membrane, incubation for 2 min at RT, centrifugation

- purified PCR products stored at – 20 °C in red box

Fold ↑

10th

Cloning DarR ORF from g-DNA

Colonies on the plates: parts 8, 7 C1

10 ml LB medium with 10 µl antibiotics, overnight culture for mini-prep

backup plates: C1, C2, C3 for part 8

Colonies on the plates: parts 8, 7 C1

Preparation of 250 ml LB broth media (stored on shelf above bench)

10 ml LB medium with 10 µl antibiotics, overnight culture for mini-prep

backup plates: C1, C2, C3 for part 8

Re-streak of E.coli clones from prtas 6 and 7 on new LB^Chloram plates

Preparation of new LB^Chloram plates (500 ml) and Preparation of 250 ml LB broth media (stored on shelf above bench)

PCR with DarR primers with different enzymes and different buffers

- dilution of primer stocks 1:20 (100 pmol à 5 pmol):

95 µl HPLC water + 5 µl primer 100 pmol

- preparation of dNTP mix à dilute stocks 1:8 (100 mM à 12.5 mM):

50 µl of dATP 100 mM

+ 50 µl of dGTP 100 mM

+ 50 µl of dTTP 100 mM

+ 50 µl of dCTP 100 mM

+ 200 µl dH₂O

è diluted primers and dNTP mix stored in red box at -20 °C

1x reaction (50 µl)

Component	Volume
Buffer (5x GC buffer or 5x HF buffer)	10 µl
dNTP mix (12.5 mM each)	2 µl
Primer fwd iGEM_34 (5 pmol)	2 µl
Primer rev iGEM_35 (5 pmol)	2 µl
Chromosomal DNA M. smegmatis	2 µl
DNA-Polymerase (Phusion or PhuS) or dH₂O for water control	1 µl
dH₂O	31 µl

- preparation of master mix for 6 reactions containing

Component	Volume
dNTP mix (12.5 mM each)	12 µl
Primer fwd iGEM_34 (5 pmol)	12 µl
Primer rev iGEM_35 (5 pmol)	12 µl
dH₂O	186 µl

- addition of template (chrom. DNA/dH₂O), polymerase and buffer individually, then addition of 37 µl master mix

è For tested combinations of buffer and Pol: see table for gel loading

- PCR protocol (cycler 7; folder “Katrin” > “iGEM” > “DarR seq”)

Step	Temperature	Time
Initial denaturation	98.5 °C	5 min
Denaturation	98.5 °C	30 s
Annealing	60 °C (T_A = T_M (≈66 °C) – 6 °C)	35 s
Elongation	72 °C	2 min (Phu needs more time than Phusion!)
Final elongation	72 °C	10 min
Hold	15 °C	∞

- 1 % Agarose-1xTAE gel

- 4 µl PCR reaction + 1 µl 5x DNA Loading Dye

- 3 µl 1 kb ladder (Quick Load)

- Run at 100 V in 1xTAE buffer

- Staining in EtBr and destaining in water

- UV detection

M

1

2

3

4

5

M

1 kb ladder

(QuickLoad)

HF

GC

HF

GC

Water controlHF

1 kb ladder

(QuickLoad)

Phusion

PhuS

è Reactions stored at - 20°C in 50 ml Falcon

Fold ↑

@@ Line 3: / Line 3: @@
 .timeline-cont{display:none}
 </style>
 <div class="tlob" id="tl_0614">
 <span class="date">14th<img src="https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png" /></span>
@@ Line 9: / Line 8: @@
 <p class="timeline-title">Observation of Back-up plates and preparation of media</p>
 <div class="timeline-cont">
-<p><b>Observation of Back-up plates</b></p>
-<ul>
+  <p class="MsoNormal"><b><span lang="EN-US">Observation of Back-up plates<o:p></o:p></span></b></p>
-	<li>Part 1, C1 and C2 probably very light pink</li>
+  <p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt;mso-list:
-	<li>Part 2, C2 and C3 became light pink, but C1 stayed white (compare with restriction analysis 13.6.13)</li>
+  l3 level1 lfo16"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:
-</ul>
+  &quot;Times New Roman&quot;;mso-bidi-font-weight:bold"><span style="mso-list:Ignore">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
-<p><b>Preparation of media for inoculation on Sunday (by Katrin Gunka)</b></p>
+  </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:
-<ul>
+  bold">Part 1, C1 and C2 probably very light pink<o:p></o:p></span></p>
-<li><u>For Plate Reader assay:</u> 4 ml LB + 4 μl Cm or Amp for C1, C2 and C3 of parts 1 – 4 and part 6, negative control DH5α in 4 ml LB</li>
+  <p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt;mso-list:
-<li><u>For Plasmid Mini-Preparation:</u> 10 ml LB + 10 μl Amp for inoculation of C2 Part 2</li>
+  l3 level1 lfo16"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:
-</ul>
+  &quot;Times New Roman&quot;;mso-bidi-font-weight:bold"><span style="mso-list:Ignore">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+  </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:
+  bold">Part 2, C2 and C3 became light pink, but C1 stayed white (compare with
+  restriction analysis <st1:chsdate isrocdate="False" islunardate="False" day="30" month="12" year="1899" w:st="on">13.6.13</st1:chsdate>)<o:p></o:p></span></p>
+  <p class="MsoNormal"><b><span lang="EN-US"><o:p>&nbsp;</o:p></span></b></p>
+  <p class="MsoNormal"><b><span lang="EN-US">Preparation of media for inoculation
+  on Sunday (by Katrin Gunka)<o:p></o:p></span></b></p>
+  <p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt;mso-list:
+  l3 level1 lfo16"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:
+  &quot;Times New Roman&quot;;mso-bidi-font-weight:bold"><span style="mso-list:Ignore">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+  </span></span></span><!--[endif]--><u><span lang="EN-US" style="mso-bidi-font-weight:
+  bold">For Plate Reader assay:</span></u><span lang="EN-US" style="mso-bidi-font-weight:
+  bold"> 4 ml LB + 4 μl Cm or Amp for C1, C2 and C3 of parts 1 – 4 and part 6,
+  negative control DH5α in 4 ml LB<o:p></o:p></span></p>
+  <p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt;mso-list:
+  l3 level1 lfo16"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:
+  &quot;Times New Roman&quot;;mso-bidi-font-weight:bold"><span style="mso-list:Ignore">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+  </span></span></span><!--[endif]--><u><span lang="EN-US" style="mso-bidi-font-weight:
+  bold">For Plasmid Mini-Preparation:</span></u><span lang="EN-US" style="mso-bidi-font-weight:bold"> 10 ml LB + 10 μl Amp for inoculation of C2
+  Part 2<b><o:p></o:p></b></span></p>
+<div class="fbutton">Fold ↑</div>
 </div>
 </div>
@@ Line 29: / Line 49: @@
 <p class="timeline-title">Test restriction of plasmids containing parts 1 – 8</p>
 <div class="timeline-cont">
-<ul>
-<li>Reactions/Master Mixes: see Excel-sheet “dropbox > iGEM > Reporter-Team > digestion system 2hour”</li>
-<li>Plasmids: parts 1 – 6 (plasmids purified on 7.6.13) and part 7, 8 (plasmids purified on 11.6.13)</li>
-<li>1x reaction with single enzyme:
-	<table>
-	<tr><th>Component</th><th>Volume</th></tr>
-	<tr><td>Enzyme</td><td>0.5μl</td></tr>
-	<tr><td>Buffer 10x</td><td>1μl</td></tr>
-	<tr><td>Plasmid DNA</td><td>200ng</td></tr>
-	<tr><td>Total volume</td><td>10μl</td></tr>
-	</table><br />
-	For double digestion:<br />
-.5 μl of each enzyme in case of EcoRI and PstI;
-          for SpeI and PstI, ratio 1:2 is recommended ? 0.5 μl SpeI
-          and 1 μl PstI used
-	</li>
+  <p class="MsoNormal"><b><span lang="EN-US">Test restriction of plasmids
-<li>Incubation at 37°C for1.5 hours</li>
+  containing parts 1 – 8<o:p></o:p></span></b></p>
-<li>Additon of 5xDNA-Loading Dye to whole reaction</li>
+  <p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt;mso-list:
-<li>Loading of 8 μl on agarose gel (~1.5 %, 1xTAE), QuickLoad 1 kb ladder as marker</li>
+  l3 level1 lfo16"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:
-<li>Run at 200 V for 1.5 h in 1xTAE</li>
+  &quot;Times New Roman&quot;;mso-bidi-font-weight:bold"><span style="mso-list:Ignore">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
-<li>EtBr staining ca. 45 min, destaining in water ca. 30 min</li>
+  </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:
-<li>UV detection</li>
+  bold">Reactions/Master Mixes: see Excel-sheet “dropbox &gt; iGEM &gt;
-</ul>
+  Reporter-Team &gt; digestion system 2hour”<o:p></o:p></span></p>
+  <p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt;mso-list:
+  l3 level1 lfo16"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:
+  &quot;Times New Roman&quot;;mso-bidi-font-weight:bold"><span style="mso-list:Ignore">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+  </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:
+  bold">Plasmids: parts 1 – 6 (plasmids purified on <st1:chsdate isrocdate="False" islunardate="False" day="30" month="12" year="1899" w:st="on">7.6.13</st1:chsdate>)
+  and part 7, 8 (plasmids purified on 11.6.13)<o:p></o:p></span></p>
+  <p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt;mso-list:
+  l3 level1 lfo16"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:
+  &quot;Times New Roman&quot;;mso-bidi-font-weight:bold"><span style="mso-list:Ignore">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+  </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:
+  bold">1x reaction with single enzyme:<o:p></o:p></span></p>
+  <table class="MsoNormalTable" border="1" cellspacing="0" cellpadding="0" style="margin-left:36.0pt;border-collapse:collapse;border:none;mso-border-alt:
+   solid black .5pt;mso-padding-alt:0cm 3.5pt 0cm 3.5pt;mso-border-insideh:
+   .5pt solid black;mso-border-insidev:.5pt solid black">
+   <tbody><tr style="mso-yfti-irow:0;mso-yfti-firstrow:yes">
+    <td width="113" valign="top" style="width:3.0cm;border:solid black 1.0pt;
+    mso-border-alt:solid black .5pt;background:#17365D;padding:0cm 3.5pt 0cm 3.5pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><b><span lang="EN-US">Component<o:p></o:p></span></b></p>
+    </td>
+    <td width="113" valign="top" style="width:3.0cm;border:solid black 1.0pt;
+    border-left:none;mso-border-left-alt:solid black .5pt;mso-border-alt:solid black .5pt;
+    background:#17365D;padding:0cm 3.5pt 0cm 3.5pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><b><span lang="EN-US">Volume<o:p></o:p></span></b></p>
+    </td>
+   </tr>
+   <tr style="mso-yfti-irow:1">
+    <td width="113" valign="top" style="width:3.0cm;border:solid black 1.0pt;
+    border-top:none;mso-border-top-alt:solid black .5pt;mso-border-alt:solid black .5pt;
+    padding:0cm 3.5pt 0cm 3.5pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><b><span lang="EN-US">Enzyme<o:p></o:p></span></b></p>
+    </td>
+    <td width="113" valign="top" style="width:3.0cm;border-top:none;border-left:
+    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
+    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">0.5 µl<o:p></o:p></span></p>
+    </td>
+   </tr>
+   <tr style="mso-yfti-irow:2">
+    <td width="113" valign="top" style="width:3.0cm;border:solid black 1.0pt;
+    border-top:none;mso-border-top-alt:solid black .5pt;mso-border-alt:solid black .5pt;
+    padding:0cm 3.5pt 0cm 3.5pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><b><span lang="EN-US">Buffer 10x<o:p></o:p></span></b></p>
+    </td>
+    <td width="113" valign="top" style="width:3.0cm;border-top:none;border-left:
+    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
+    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">1 µl<o:p></o:p></span></p>
+    </td>
+   </tr>
+   <tr style="mso-yfti-irow:3">
+    <td width="113" valign="top" style="width:3.0cm;border:solid black 1.0pt;
+    border-top:none;mso-border-top-alt:solid black .5pt;mso-border-alt:solid black .5pt;
+    padding:0cm 3.5pt 0cm 3.5pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><b><span lang="EN-US">Plasmid DNA<o:p></o:p></span></b></p>
+    </td>
+    <td width="113" valign="top" style="width:3.0cm;border-top:none;border-left:
+    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
+    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">200 ng<o:p></o:p></span></p>
+    </td>
+   </tr>
+   <tr style="mso-yfti-irow:4;mso-yfti-lastrow:yes">
+    <td width="113" valign="top" style="width:3.0cm;border:solid black 1.0pt;
+    border-top:none;mso-border-top-alt:solid black .5pt;mso-border-alt:solid black .5pt;
+    background:#8DB3E2;padding:0cm 3.5pt 0cm 3.5pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><b><span lang="EN-US">Total volume<o:p></o:p></span></b></p>
+    </td>
+    <td width="113" valign="top" style="width:3.0cm;border-top:none;border-left:
+    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
+    mso-border-alt:solid black .5pt;background:#8DB3E2;padding:0cm 3.5pt 0cm 3.5pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">10 µl<o:p></o:p></span></p>
+    </td>
+   </tr>
+  </tbody></table>
+  <p class="MsoNormal" style="margin-left:54.0pt;text-indent:-18.0pt;mso-list:
+  l1 level1 lfo14"><!--[if !supportLists]--><span lang="EN-US" style="font-family:
+  Wingdings;mso-fareast-font-family:Wingdings;mso-bidi-font-family:Wingdings;
+  mso-bidi-font-weight:bold"><span style="mso-list:Ignore">è<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:bold">For double digestion:<o:p></o:p></span></p>
+  <p class="MsoNormal"><span lang="EN-US" style="mso-bidi-font-weight:bold"><span style="mso-spacerun:yes">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+  </span>0.5 µl of each enzyme in case of EcoRI and PstI;<o:p></o:p></span></p>
+  <p class="MsoNormal"><span lang="EN-US" style="mso-bidi-font-weight:bold"><span style="mso-spacerun:yes">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+  </span>for SpeI and PstI, ratio 1:2 is recommended </span><span lang="EN-US" style="font-family:Wingdings;mso-ascii-font-family:&quot;Times New Roman&quot;;
+  mso-hansi-font-family:&quot;Times New Roman&quot;;mso-char-type:symbol;mso-symbol-font-family:
+  Wingdings;mso-bidi-font-weight:bold"><span style="mso-char-type:symbol;
+  mso-symbol-font-family:Wingdings">à</span></span><span lang="EN-US" style="mso-bidi-font-weight:bold"> 0.5 µl SpeI and 1 µl PstI used<o:p></o:p></span></p>
+  <p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt;mso-list:
+  l3 level1 lfo16"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:
+  &quot;Times New Roman&quot;;mso-bidi-font-weight:bold"><span style="mso-list:Ignore">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+  </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:
+  bold">Incubation at 37°C for1.5 hours<o:p></o:p></span></p>
+  <p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt;mso-list:
+  l3 level1 lfo16"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:
+  &quot;Times New Roman&quot;;mso-bidi-font-weight:bold"><span style="mso-list:Ignore">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+  </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:
+  bold">Additon of 5xDNA-Loading Dye to whole reaction<o:p></o:p></span></p>
+  <p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt;mso-list:
+  l3 level1 lfo16"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:
+  &quot;Times New Roman&quot;;mso-bidi-font-weight:bold"><span style="mso-list:Ignore">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+  </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:
+  bold">Loading of 8 µl on agarose gel (~1.5 %, 1xTAE), QuickLoad 1 kb ladder
+  as marker<o:p></o:p></span></p>
+  <p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt;mso-list:
+  l3 level1 lfo16"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:
+  &quot;Times New Roman&quot;;mso-bidi-font-weight:bold"><span style="mso-list:Ignore">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+  </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:
+  bold">Run at 200 V for 1.5 h in 1xTAE<o:p></o:p></span></p>
+  <p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt;mso-list:
+  l3 level1 lfo16"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:
+  &quot;Times New Roman&quot;;mso-bidi-font-weight:bold"><span style="mso-list:Ignore">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+  </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:
+  bold">EtBr staining ca. 45 min, destaining in water ca. 30 min<o:p></o:p></span></p>
+  <p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt;mso-list:
+  l3 level1 lfo16"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:
+  &quot;Times New Roman&quot;;mso-bidi-font-weight:bold"><span style="mso-list:Ignore">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+  </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:
+  bold">UV detection<o:p></o:p></span></p>
+  <p class="MsoNormal"><b><span lang="EN-US"><o:p>&nbsp;</o:p></span></b></p>
+  <p class="MsoNormal"><b><span lang="EN-US">Expected Fragments:<o:p></o:p></span></b></p>
+  <table class="MsoNormalTable" border="1" cellspacing="0" cellpadding="0" style="border-collapse:collapse;border:none;mso-border-alt:solid black .5pt;
+   mso-padding-alt:0cm 5.4pt 0cm 5.4pt;mso-border-insideh:.5pt solid black;
+   mso-border-insidev:.5pt solid black">
+   <tbody><tr style="mso-yfti-irow:0;mso-yfti-firstrow:yes">
+    <td width="125" style="width:93.75pt;border:solid black 1.0pt;mso-border-alt:
+    solid black .5pt;padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><b style="mso-bidi-font-weight:normal"><span lang="EN-US" style="mso-bidi-font-size:
+.0pt">Part no.<o:p></o:p></span></b></p>
+    </td>
+    <td width="144" style="width:107.85pt;border:solid black 1.0pt;border-left:
+    none;mso-border-left-alt:solid black .5pt;mso-border-alt:solid black .5pt;
+    padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><b style="mso-bidi-font-weight:normal"><span lang="EN-US" style="mso-bidi-font-size:
+.0pt">Fragments for PstI and SpeI (bp)<o:p></o:p></span></b></p>
+    </td>
+    <td width="193" style="width:144.7pt;border:solid black 1.0pt;border-left:
+    none;mso-border-left-alt:solid black .5pt;mso-border-alt:solid black .5pt;
+    padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><b style="mso-bidi-font-weight:normal"><span lang="EN-US" style="mso-bidi-font-size:
+.0pt">Plasmid size (linearization with PstI or SpeI)(bp)<o:p></o:p></span></b></p>
+    </td>
+   </tr>
+   <tr style="mso-yfti-irow:1;page-break-inside:avoid">
+    <td width="125" style="width:93.75pt;border:solid black 1.0pt;border-top:
+    none;mso-border-top-alt:solid black .5pt;mso-border-alt:solid black .5pt;
+    padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-size:12.0pt">1<o:p></o:p></span></p>
+    </td>
+    <td width="144" rowspan="4" style="width:107.85pt;border-top:none;border-left:
+    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
+    mso-border-alt:solid black .5pt;padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-size:12.0pt">~900 + 2000 <o:p></o:p></span></p>
+    </td>
+    <td width="193" rowspan="4" style="width:144.7pt;border-top:none;border-left:
+    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
+    mso-border-alt:solid black .5pt;padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-size:12.0pt">2948<o:p></o:p></span></p>
+    </td>
+   </tr>
+   <tr style="mso-yfti-irow:2;page-break-inside:avoid">
+    <td width="125" style="width:93.75pt;border:solid black 1.0pt;border-top:
+    none;mso-border-top-alt:solid black .5pt;mso-border-alt:solid black .5pt;
+    padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-size:12.0pt">2<o:p></o:p></span></p>
+    </td>
+   </tr>
+   <tr style="mso-yfti-irow:3;page-break-inside:avoid">
+    <td width="125" style="width:93.75pt;border:solid black 1.0pt;border-top:
+    none;mso-border-top-alt:solid black .5pt;mso-border-alt:solid black .5pt;
+    padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-size:12.0pt">3<o:p></o:p></span></p>
+    </td>
+   </tr>
+   <tr style="mso-yfti-irow:4;page-break-inside:avoid">
+    <td width="125" style="width:93.75pt;border:solid black 1.0pt;border-top:
+    none;mso-border-top-alt:solid black .5pt;mso-border-alt:solid black .5pt;
+    padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-size:12.0pt">4<o:p></o:p></span></p>
+    </td>
+   </tr>
+   <tr style="mso-yfti-irow:5">
+    <td width="125" style="width:93.75pt;border:solid black 1.0pt;border-top:
+    none;mso-border-top-alt:solid black .5pt;mso-border-alt:solid black .5pt;
+    padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><b style="mso-bidi-font-weight:normal"><span lang="EN-US" style="mso-bidi-font-size:
+.0pt">Part no.</span></b><span lang="EN-US" style="mso-bidi-font-size:12.0pt"><o:p></o:p></span></p>
+    </td>
+    <td width="144" style="width:107.85pt;border-top:none;border-left:none;
+    border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;mso-border-top-alt:
+    solid black .5pt;mso-border-left-alt:solid black .5pt;mso-border-alt:solid black .5pt;
+    padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><b style="mso-bidi-font-weight:normal"><span lang="EN-US" style="mso-bidi-font-size:
+.0pt">Fragments for PstI and EcoRI(bp)</span></b><span lang="EN-US" style="mso-bidi-font-size:12.0pt"><o:p></o:p></span></p>
+    </td>
+    <td width="193" style="width:144.7pt;border-top:none;border-left:none;
+    border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;mso-border-top-alt:
+    solid black .5pt;mso-border-left-alt:solid black .5pt;mso-border-alt:solid black .5pt;
+    padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><b style="mso-bidi-font-weight:normal"><span lang="EN-US" style="mso-bidi-font-size:
+.0pt">Plasmid size (linearization with PstI or EcoRI)(bp)</span></b><span lang="EN-US" style="mso-bidi-font-size:12.0pt"><o:p></o:p></span></p>
+    </td>
+   </tr>
+   <tr style="mso-yfti-irow:6">
+    <td width="125" style="width:93.75pt;border:solid black 1.0pt;border-top:
+    none;mso-border-top-alt:solid black .5pt;mso-border-alt:solid black .5pt;
+    padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-size:12.0pt">5<o:p></o:p></span></p>
+    </td>
+    <td width="337" colspan="2" style="width:252.55pt;border-top:none;border-left:
+    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
+    mso-border-alt:solid black .5pt;padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-size:12.0pt">???<o:p></o:p></span></p>
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-size:12.0pt">(if RFP gene </span><span lang="EN-US" style="mso-bidi-font-size:12.0pt;font-family:Wingdings;mso-ascii-font-family:
+    &quot;Times New Roman&quot;;mso-hansi-font-family:&quot;Times New Roman&quot;;mso-char-type:
+    symbol;mso-symbol-font-family:Wingdings"><span style="mso-char-type:symbol;
+    mso-symbol-font-family:Wingdings">à</span></span><span lang="EN-US" style="mso-bidi-font-size:12.0pt"> same pattern as expected for parts 1 –
+; if no RPF gene </span><span lang="EN-US" style="mso-bidi-font-size:12.0pt;
+    font-family:Wingdings;mso-ascii-font-family:&quot;Times New Roman&quot;;mso-hansi-font-family:
+    &quot;Times New Roman&quot;;mso-char-type:symbol;mso-symbol-font-family:Wingdings"><span style="mso-char-type:symbol;mso-symbol-font-family:Wingdings">à</span></span><span lang="EN-US" style="mso-bidi-font-size:12.0pt"> same pattern as expected for
+    part 8)<o:p></o:p></span></p>
+    </td>
+   </tr>
+   <tr style="mso-yfti-irow:7">
+    <td width="125" style="width:93.75pt;border:solid black 1.0pt;border-top:
+    none;mso-border-top-alt:solid black .5pt;mso-border-alt:solid black .5pt;
+    padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-size:12.0pt">6<o:p></o:p></span></p>
+    </td>
+    <td width="144" style="width:107.85pt;border-top:none;border-left:none;
+    border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;mso-border-top-alt:
+    solid black .5pt;mso-border-left-alt:solid black .5pt;mso-border-alt:solid black .5pt;
+    padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-size:12.0pt">917<o:p></o:p></span></p>
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-size:12.0pt">2029<o:p></o:p></span></p>
+    </td>
+    <td width="193" style="width:144.7pt;border-top:none;border-left:none;
+    border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;mso-border-top-alt:
+    solid black .5pt;mso-border-left-alt:solid black .5pt;mso-border-alt:solid black .5pt;
+    padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-size:12.0pt">2946<o:p></o:p></span></p>
+    </td>
+   </tr>
+   <tr style="mso-yfti-irow:8">
+    <td width="125" style="width:93.75pt;border:solid black 1.0pt;border-top:
+    none;mso-border-top-alt:solid black .5pt;mso-border-alt:solid black .5pt;
+    padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-size:12.0pt">7<o:p></o:p></span></p>
+    </td>
+    <td width="144" style="width:107.85pt;border-top:none;border-left:none;
+    border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;mso-border-top-alt:
+    solid black .5pt;mso-border-left-alt:solid black .5pt;mso-border-alt:solid black .5pt;
+    padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-size:12.0pt">140<o:p></o:p></span></p>
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-size:12.0pt">2050<o:p></o:p></span></p>
+    </td>
+    <td width="193" style="width:144.7pt;border-top:none;border-left:none;
+    border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;mso-border-top-alt:
+    solid black .5pt;mso-border-left-alt:solid black .5pt;mso-border-alt:solid black .5pt;
+    padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-size:12.0pt">2199<o:p></o:p></span></p>
+    </td>
+   </tr>
+   <tr style="mso-yfti-irow:9;mso-yfti-lastrow:yes">
+    <td width="125" style="width:93.75pt;border:solid black 1.0pt;border-top:
+    none;mso-border-top-alt:solid black .5pt;mso-border-alt:solid black .5pt;
+    padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-size:12.0pt">8<o:p></o:p></span></p>
+    </td>
+    <td width="144" style="width:107.85pt;border-top:none;border-left:none;
+    border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;mso-border-top-alt:
+    solid black .5pt;mso-border-left-alt:solid black .5pt;mso-border-alt:solid black .5pt;
+    padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-size:12.0pt">53<o:p></o:p></span></p>
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-size:12.0pt">2038<o:p></o:p></span></p>
+    </td>
+    <td width="193" style="width:144.7pt;border-top:none;border-left:none;
+    border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;mso-border-top-alt:
+    solid black .5pt;mso-border-left-alt:solid black .5pt;mso-border-alt:solid black .5pt;
+    padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-size:12.0pt">2091<o:p></o:p></span></p>
+    </td>
+   </tr>
+  </tbody></table>
+  <p class="MsoNormal"><b><span lang="EN-US"><o:p>&nbsp;</o:p></span></b></p>
+  <p class="MsoNormal"><b><span lang="EN-US">Gel: </span></b><span lang="DE" style="mso-ansi-language:DE;mso-bidi-font-weight:bold">See ppt-file “dropbox
+  &gt; iGEM &gt; Reporter-Team &gt; Geldoc“<o:p></o:p></span></p>
+  <p class="MsoNormal"><span lang="DE" style="mso-ansi-language:DE;mso-fareast-language:
+  DE"><!--[if gte vml 1]><v:shape id="Objekt_x0020_2" o:spid="_x0000_i1026"
+   type="#_x0000_t75" style='width:282.75pt;height:204pt;
+   mso-position-horizontal-relative:page;mso-position-vertical-relative:page'>
+   <v:imagedata src="part1.files/image003.png" o:title="" croptop="-1230f"
+    cropbottom="-168f" cropleft="-598f"/>
+  </v:shape><![endif]--><!--[if !vml]--> <img src="https://static.igem.org/mediawiki/2013/8/8f/Goe-13.6.13-RT.png" width="500px" v:shapes="Objekt_x0020_2"><!--[endif]--></span><span lang="EN-US" style="mso-bidi-font-weight:bold"><o:p></o:p></span></p>
+  <p class="MsoNormal"><b><span lang="EN-US">Conclusions:<o:p></o:p></span></b></p>
+  <p class="MsoNormal" style="margin-left:54.0pt;text-indent:-18.0pt;mso-list:
+  l1 level1 lfo14"><!--[if !supportLists]--><span lang="EN-US" style="font-family:
+  Wingdings;mso-fareast-font-family:Wingdings;mso-bidi-font-family:Wingdings;
+  mso-bidi-font-weight:bold"><span style="mso-list:Ignore">è<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><!--[endif]--><b><span lang="EN-US">Partial digestion</span></b><span lang="EN-US" style="mso-bidi-font-weight:
+  bold"> (next time: longer incubation time, less plasmid…)<o:p></o:p></span></p>
+  <p class="MsoNormal" style="margin-left:54.0pt;text-indent:-18.0pt;mso-list:
+  l1 level1 lfo14"><!--[if !supportLists]--><span lang="EN-US" style="font-family:
+  Wingdings;mso-fareast-font-family:Wingdings;mso-bidi-font-family:Wingdings;
+  mso-bidi-font-weight:bold"><span style="mso-list:Ignore">è<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:bold">For <b>parts 1, 3, 4, 6, 7 and 8</b>,
+  the expected bands were observed (<b>ok</b>)<o:p></o:p></span></p>
+  <p class="MsoNormal" style="margin-left:54.0pt;text-indent:-18.0pt;mso-list:
+  l1 level1 lfo14"><!--[if !supportLists]--><span lang="EN-US" style="font-family:
+  Wingdings;mso-fareast-font-family:Wingdings;mso-bidi-font-family:Wingdings;
+  mso-bidi-font-weight:bold"><span style="mso-list:Ignore">è<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:bold">Band pattern of <b>part 5 </b>resembles
+  that expected for parts 1 – 4 </span><span lang="EN-US" style="font-family:
+  Wingdings;mso-ascii-font-family:&quot;Times New Roman&quot;;mso-hansi-font-family:&quot;Times New Roman&quot;;
+  mso-char-type:symbol;mso-symbol-font-family:Wingdings;mso-bidi-font-weight:
+  bold"><span style="mso-char-type:symbol;mso-symbol-font-family:Wingdings">à</span></span><span lang="EN-US" style="mso-bidi-font-weight:bold"> this plasmid <b>contains RFP
+  gene</b><o:p></o:p></span></p>
+  <p class="MsoNormal" style="margin-left:54.0pt;text-indent:-18.0pt;mso-list:
+  l1 level1 lfo14"><!--[if !supportLists]--><span lang="EN-US" style="font-family:
+  Wingdings;mso-fareast-font-family:Wingdings;mso-bidi-font-family:Wingdings;
+  mso-bidi-font-weight:bold"><span style="mso-list:Ignore">è<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:bold">In case of <b>part 2, SpeI was
+  unable to cut the plasmid</b> </span><span lang="EN-US" style="font-family:
+  Wingdings;mso-ascii-font-family:&quot;Times New Roman&quot;;mso-hansi-font-family:&quot;Times New Roman&quot;;
+  mso-char-type:symbol;mso-symbol-font-family:Wingdings;mso-bidi-font-weight:
+  bold"><span style="mso-char-type:symbol;mso-symbol-font-family:Wingdings">à</span></span><span lang="EN-US" style="mso-bidi-font-weight:bold"> no cleavage for SpeI single
+  digest + linearization for SpeI/PstI double digest </span><span lang="EN-US" style="font-family:Wingdings;mso-ascii-font-family:&quot;Times New Roman&quot;;
+  mso-hansi-font-family:&quot;Times New Roman&quot;;mso-char-type:symbol;mso-symbol-font-family:
+  Wingdings;mso-bidi-font-weight:bold"><span style="mso-char-type:symbol;
+  mso-symbol-font-family:Wingdings">à</span></span><span lang="EN-US" style="mso-bidi-font-weight:bold"> plasmid contains probably something else
+  (SpeI/XbaI scar at actual SpeI site?)<o:p></o:p></span></p>
+  <p class="MsoNormal" style="margin-left:54.0pt;text-indent:-18.0pt;mso-list:
+  l1 level1 lfo14"><!--[if !supportLists]--><span lang="EN-US" style="font-family:
+  Wingdings;mso-fareast-font-family:Wingdings;mso-bidi-font-family:Wingdings;
+  mso-bidi-font-weight:bold"><span style="mso-list:Ignore">è<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><!--[endif]--><b><span lang="EN-US">For cloning: terminator (part 7) and strong RBS (part 8) might be
+  difficult to extract from the gel, since they are very short </span></b><b><span lang="EN-US" style="font-family:Wingdings;mso-ascii-font-family:&quot;Times New Roman&quot;;
+  mso-hansi-font-family:&quot;Times New Roman&quot;;mso-char-type:symbol;mso-symbol-font-family:
+  Wingdings"><span style="mso-char-type:symbol;mso-symbol-font-family:Wingdings">à</span></span><span lang="EN-US"> think of other cloning strategy without gel extraction (e.g.
+  “play” with resistances of plasmid backbones to digest backbone after cutting
+  out part…)<o:p></o:p></span></b></p>
+  <p class="MsoNormal"><b><span lang="EN-US"><o:p>&nbsp;</o:p></span></b></p>
+  <p class="MsoNormal"><b><span lang="EN-US">Preparation of DarR PCR samples for
+  sequencing<o:p></o:p></span></b></p>
+  <p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt;mso-list:
+  l3 level1 lfo16"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:
+  &quot;Times New Roman&quot;;mso-bidi-font-weight:bold"><span style="mso-list:Ignore">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+  </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:
+  bold">4 µl of DarR seq PCR product of reaction 1 + 1 µl of iGEM_34 (fwd) or
+  iGEM_35 (rev)<o:p></o:p></span></p>
+  <p class="MsoNormal"><span lang="DE" style="mso-ansi-language:DE;mso-bidi-font-weight:
+  bold">Tubes:<o:p></o:p></span></p>
+  <p class="MsoNormal"><span lang="DE" style="mso-ansi-language:DE;mso-bidi-font-weight:
+  bold">kgun_1 </span><span lang="EN-US" style="font-family:Wingdings;mso-ascii-font-family:
+  &quot;Times New Roman&quot;;mso-hansi-font-family:&quot;Times New Roman&quot;;mso-char-type:symbol;
+  mso-symbol-font-family:Wingdings;mso-bidi-font-weight:bold"><span style="mso-char-type:symbol;mso-symbol-font-family:Wingdings">à</span></span><span lang="DE" style="mso-ansi-language:DE;mso-bidi-font-weight:bold"> iGEM_34<o:p></o:p></span></p>
+  <p class="MsoNormal"><span lang="DE" style="mso-ansi-language:DE;mso-bidi-font-weight:
+  bold">kgun_2 </span><span lang="EN-US" style="font-family:Wingdings;mso-ascii-font-family:
+  &quot;Times New Roman&quot;;mso-hansi-font-family:&quot;Times New Roman&quot;;mso-char-type:symbol;
+  mso-symbol-font-family:Wingdings;mso-bidi-font-weight:bold"><span style="mso-char-type:symbol;mso-symbol-font-family:Wingdings">à</span></span><span lang="DE" style="mso-ansi-language:DE;mso-bidi-font-weight:bold"> iGEM_35<o:p></o:p></span></p>
+  <p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt;mso-list:
+  l3 level1 lfo16"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:
+  &quot;Times New Roman&quot;;mso-bidi-font-weight:bold"><span style="mso-list:Ignore">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+  </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:
+  bold">Sequencing at G2L<b><o:p></o:p></b></span></p>
-<p><b>Expected Fragments:</b></p>
+<div class="fbutton">Fold ↑</div>
-<table cellspacing="0" border="1">
-<tr><th>Part No.</th><th>Fragments for PstI and SpeI (bp)</th><th>Plasmid size <br />(linearization with PstI or SpeI)(bp)</th></tr>
-<tr><td>1</td><td rowspan="4">~900+2000</td><td rowspan="4">2948</td></tr>
-<tr><td>2</td></tr>
-<tr><td>3</td></tr>
-<tr><td>4</td></tr>
-<tr><th>Part no.</th><th>Fragments for PstI and EcoRI(bp)</th><th>Plasmid size <br />(linearization with PstI or EcoRI)(bp)</th></tr>
-<tr><td>5</td><td colspan="2">(if RFP gene :same pattern as expected for parts 1 – 4;<br /> if no RPF gene :same pattern as expected for part 8)</td></tr>
-<tr><td>6</td><td>917+2029</td><td>2946</td></tr>
-<tr><td>7</td><td>140+2050</td><td>2199</td></tr>
-<tr><td>8</td><td>53+2038</td><td>2091</td></tr>
-</table>
-<p><b>Gel:See ppt-file “dropbox > iGEM > Reporter-Team > Geldoc“</b></p>
- <img src="https://static.igem.org/mediawiki/2013/8/8f/Goe-13.6.13-RT.png" width="500px" />
- <p><b>Conclusions:</b></p>
-<ul>
-<li><b>Partial digestion </b>(next time: longer incubation time, less plasmid…)</li>
-<li>For <b>parts 1, 3, 4, 6, 7 and 8, </b>the expected bands were observed (ok)</li>
-<li>Band pattern of <b>part 5</b> resembles that expected for parts 1 – 4 -> this plasmid <b>contains RFP gene</b></li>
-<li>In case of <b> part 2, SpeI was unable to cut the plasmid</b> -> no cleavage for SpeI single digest + linearization for SpeI/PstI double digest ? plasmid contains probably something else (SpeI/XbaI scar at actual SpeI site?)</li>
-<li><b>For cloning: terminator (part 7) and strong RBS (part 8) might be difficult to extract from the gel, since they are very short ? think of other cloning strategy without gel extraction (e.g. “play” with resistances of plasmid backbones to digest backbone after cutting out part…)</b></li>
-</ul>
-<p><b>Preparation of DarR PCR samples for sequencing</b></p>
-<p>4 μl of DarR seq PCR product of reaction 1 + 1 μl of iGEM_34 (fwd) or iGEM_35 (rev)</p>
-Tubes:<ul>
-<li>kgun_1 -> iGEM_34</li>
-<li>kgun_2 -> iGEM_35</li>
-</ul>
-<p>Sequencing at G2L</p>
 </div>
 </div>
 </div>
+  <div class="tlob" id="tl_0611">
+<span class="date">11th<img src="https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png"></span>
+<div class="cont">
+<p class="timeline-title" title="Click to fold/extend">Miniprep of Part7,8 and DarR sequencing PCR clean-up</p>
+<div class="timeline-cont" style="display: block;">
+  <p class="MsoNormal"><b><span lang="EN-US">Cryo-Stocks of <i style="mso-bidi-font-style:
+  normal">E. coli</i> transfomants (8, 7 C1)<o:p></o:p></span></b></p>
+  <p class="MsoNormal"><span lang="EN-US" style="mso-bidi-font-weight:bold">Stored
+  in -70 °C (red box)<o:p></o:p></span></p>
+  <p class="MsoNormal"><span lang="EN-US" style="mso-bidi-font-weight:bold"><o:p>&nbsp;</o:p></span></p>
+  <p class="MsoNormal"><b><span lang="EN-US">Plasmid Mini-Preparation of parts 8, 7
+  C1<o:p></o:p></span></b></p>
+  <p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt;mso-list:
+  l5 level1 lfo6"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:
+  &quot;Times New Roman&quot;;mso-bidi-font-weight:bold"><span style="mso-list:Ignore">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+  </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:
+  bold">harvesting of ca. 5 ml pellets in 2 ml Epis for future Plasmid
+  Mini-Preparation </span><span lang="EN-US" style="font-family:Wingdings;
+  mso-ascii-font-family:&quot;Times New Roman&quot;;mso-hansi-font-family:&quot;Times New Roman&quot;;
+  mso-char-type:symbol;mso-symbol-font-family:Wingdings;mso-bidi-font-weight:
+  bold"><span style="mso-char-type:symbol;mso-symbol-font-family:Wingdings">à</span></span><span lang="EN-US" style="mso-bidi-font-weight:bold"> stored at – 20 °C in red box<o:p></o:p></span></p>
+  <p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt;mso-list:
+  l5 level1 lfo6"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:
+  &quot;Times New Roman&quot;;mso-bidi-font-weight:bold"><span style="mso-list:Ignore">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+  </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:
+  bold">harvesting of remaining culture for today’s prep<o:p></o:p></span></p>
+  <p class="MsoNormal" style="margin-left:54.0pt;text-indent:-18.0pt;mso-list:
+  l1 level1 lfo7"><!--[if !supportLists]--><span lang="EN-US" style="font-family:
+  Wingdings;mso-fareast-font-family:Wingdings;mso-bidi-font-family:Wingdings;
+  mso-bidi-font-weight:bold"><span style="mso-list:Ignore">è<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:bold">Performed as on <st1:chsdate year="1899" month="12" day="30" islunardate="False" isrocdate="False" w:st="on">7.6.13</st1:chsdate><o:p></o:p></span></p>
+  <p class="MsoNormal"><span lang="EN-US" style="mso-bidi-font-weight:bold">NanoDrop
+  – Plasmid concentrations<o:p></o:p></span></p>
+  <table class="MsoNormalTable" border="1" cellspacing="0" cellpadding="0" style="border-collapse:collapse;border:none;mso-border-alt:solid black .5pt;
+   mso-padding-alt:0cm 3.5pt 0cm 3.5pt;mso-border-insideh:.5pt solid black;
+   mso-border-insidev:.5pt solid black">
+   <tbody><tr style="mso-yfti-irow:0;mso-yfti-firstrow:yes">
+    <td width="100" valign="top" style="width:74.7pt;border:solid black 1.0pt;
+    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><b><span lang="EN-US">Part no.<o:p></o:p></span></b></p>
+    </td>
+    <td width="100" valign="top" style="width:74.75pt;border:solid black 1.0pt;
+    border-left:none;mso-border-left-alt:solid black .5pt;mso-border-alt:solid black .5pt;
+    padding:0cm 3.5pt 0cm 3.5pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><b><span lang="EN-US">c(DNA)<o:p></o:p></span></b></p>
+    <p class="MsoNormal" align="center" style="text-align:center"><b><span lang="EN-US" style="font-family:Calibri">[</span><span lang="EN-US">ng/µl]<o:p></o:p></span></b></p>
+    </td>
+    <td width="100" valign="top" style="width:74.75pt;border:solid black 1.0pt;
+    border-left:none;mso-border-left-alt:solid black .5pt;mso-border-alt:solid black .5pt;
+    padding:0cm 3.5pt 0cm 3.5pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><b><span lang="EN-US">A<sub>260</sub>/A<sub>280</sub><o:p></o:p></span></b></p>
+    </td>
+    <td width="100" valign="top" style="width:74.75pt;border:solid black 1.0pt;
+    border-left:none;mso-border-left-alt:solid black .5pt;mso-border-alt:solid black .5pt;
+    padding:0cm 3.5pt 0cm 3.5pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><b><span lang="EN-US">A<sub>260</sub>/A<sub>230</sub><o:p></o:p></span></b></p>
+    </td>
+   </tr>
+   <tr style="mso-yfti-irow:1">
+    <td width="100" valign="top" style="width:74.7pt;border:solid black 1.0pt;
+    border-top:none;mso-border-top-alt:solid black .5pt;mso-border-alt:solid black .5pt;
+    padding:0cm 3.5pt 0cm 3.5pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">7<o:p></o:p></span></p>
+    </td>
+    <td width="100" valign="top" style="width:74.75pt;border-top:none;border-left:
+    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
+    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">67.0<o:p></o:p></span></p>
+    </td>
+    <td width="100" valign="top" style="width:74.75pt;border-top:none;border-left:
+    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
+    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">1.98<o:p></o:p></span></p>
+    </td>
+    <td width="100" valign="top" style="width:74.75pt;border-top:none;border-left:
+    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
+    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">2.25<o:p></o:p></span></p>
+    </td>
+   </tr>
+   <tr style="mso-yfti-irow:2;mso-yfti-lastrow:yes">
+    <td width="100" valign="top" style="width:74.7pt;border:solid black 1.0pt;
+    border-top:none;mso-border-top-alt:solid black .5pt;mso-border-alt:solid black .5pt;
+    padding:0cm 3.5pt 0cm 3.5pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">8<o:p></o:p></span></p>
+    </td>
+    <td width="100" valign="top" style="width:74.75pt;border-top:none;border-left:
+    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
+    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">62.7<o:p></o:p></span></p>
+    </td>
+    <td width="100" valign="top" style="width:74.75pt;border-top:none;border-left:
+    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
+    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">1.89<o:p></o:p></span></p>
+    </td>
+    <td width="100" valign="top" style="width:74.75pt;border-top:none;border-left:
+    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
+    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">2.08<o:p></o:p></span></p>
+    </td>
+   </tr>
+  </tbody></table>
+  <p class="MsoNormal"><span lang="EN-US" style="mso-bidi-font-weight:bold"><o:p>&nbsp;</o:p></span></p>
+  <p class="MsoNormal"><b><span lang="EN-US">DarR seq PCR purification<o:p></o:p></span></b></p>
+  <p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt;mso-list:
+  l3 level1 lfo8"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:
+  &quot;Times New Roman&quot;;mso-bidi-font-weight:bold"><span style="mso-list:Ignore">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+  </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:
+  bold">for DarR seq PCR reactions 1 - 4<o:p></o:p></span></p>
+  <p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt;mso-list:
+  l3 level1 lfo8"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:
+  &quot;Times New Roman&quot;;mso-bidi-font-weight:bold"><span style="mso-list:Ignore">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+  </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:
+  bold">with QIAquick PCR purification Kit (Qiagen), according to quick start
+  manual<o:p></o:p></span></p>
+  <p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt;mso-list:
+  l3 level1 lfo8"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:
+  &quot;Times New Roman&quot;;mso-bidi-font-weight:bold"><span style="mso-list:Ignore">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+  </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:
+  bold">ca. 50 µl of PCR reaction + 250 µl of PB buffer<o:p></o:p></span></p>
+  <p class="MsoNormal" style="margin-left:36.0pt"><span lang="EN-US" style="mso-bidi-font-weight:bold">reactions 2 and 4 turned violet </span><span lang="EN-US" style="font-family:Wingdings;mso-ascii-font-family:&quot;Times New Roman&quot;;
+  mso-hansi-font-family:&quot;Times New Roman&quot;;mso-char-type:symbol;mso-symbol-font-family:
+  Wingdings;mso-bidi-font-weight:bold"><span style="mso-char-type:symbol;
+  mso-symbol-font-family:Wingdings">à</span></span><span lang="EN-US" style="mso-bidi-font-weight:bold"> addition of 10 µl NaAc 3.3 M, pH = 5.0<o:p></o:p></span></p>
+  <p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt;mso-list:
+  l3 level1 lfo8"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:
+  &quot;Times New Roman&quot;;mso-bidi-font-weight:bold"><span style="mso-list:Ignore">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+  </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:
+  bold">elution: 30 µl pre-warmed HPLC water directly applied to membrane,
+  incubation for 2 min at RT, centrifugation<o:p></o:p></span></p>
+  <p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt;mso-list:
+  l3 level1 lfo8"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:
+  &quot;Times New Roman&quot;;mso-bidi-font-weight:bold"><span style="mso-list:Ignore">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+  </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:
+  bold">purified PCR products stored at – 20 °C in red box<o:p></o:p></span></p>
+<div class="fbutton">Fold ↑</div>
+</div>
+</div>
+</div>
-<div class="tlob" id="tl_0611">
-<span class="date">11th<img src="https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png" /></span>
+<div class="tlob" id="tl_0610">
+<span class="date">10th<img src="https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png"></span>
 <div class="cont">
-<p class="timeline-title">Miniprep of Part7,8 and DarR sequencing PCR clean-up</p>
+<p class="timeline-title" title="Click to fold/extend">Cloning DarR ORF from g-DNA</p>
-<div class="timeline-cont">
+<div class="timeline-cont" style="display: block;">
-<b>Cryo-Stocks of E. coli transfomants (8, 7 Cm)</b>
-<p>stored in -70 °C (red box)</p>
+  <p class="MsoNormal"><b><span lang="EN-US">Colonies on the plates: parts 8, 7 C1</span></b><span lang="EN-US" style="mso-bidi-font-weight:bold"><o:p></o:p></span></p>
+  <p class="MsoNormal"><span lang="EN-US" style="mso-bidi-font-weight:bold">10 ml
+  LB medium with 10</span><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:
+  bold"> </span><span lang="EN-US" style="font-size:10.0pt;color:#262626;
+  mso-font-kerning:0pt;mso-fareast-language:DE">µl</span><span lang="EN-US" style="font-size:12.0pt;font-family:Verdana;mso-bidi-font-family:Verdana;
+  color:#262626;mso-font-kerning:0pt;mso-fareast-language:DE"> </span><span lang="EN-US" style="mso-bidi-font-weight:bold">antibiotics, overnight culture
+  for mini-prep<o:p></o:p></span></p>
+  <p class="MsoNormal"><span lang="EN-US" style="mso-bidi-font-weight:bold">backup
+  plates: C1, C2, C3 for part 8<o:p></o:p></span></p>
+  <p class="MsoNormal"><b><span lang="EN-US"><o:p>&nbsp;</o:p></span></b></p>
+  <p class="MsoNormal"><b><span lang="EN-US">Colonies on the plates: parts 8, 7 C1</span></b><span lang="EN-US" style="mso-bidi-font-weight:bold"><o:p></o:p></span></p>
+  <p class="MsoNormal"><span lang="EN-US" style="mso-bidi-font-weight:bold">Preparation
+  of 250 ml LB broth media (stored on shelf above bench)<o:p></o:p></span></p>
+  <p class="MsoNormal"><span lang="EN-US" style="mso-bidi-font-weight:bold">10 ml
+  LB medium with 10</span><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:
+  bold"> </span><span lang="EN-US" style="font-size:10.0pt;color:#262626;
+  mso-font-kerning:0pt;mso-fareast-language:DE">µl</span><span lang="EN-US" style="font-size:12.0pt;font-family:Verdana;mso-bidi-font-family:Verdana;
+  color:#262626;mso-font-kerning:0pt;mso-fareast-language:DE"> </span><span lang="EN-US" style="mso-bidi-font-weight:bold">antibiotics, overnight culture
+  for mini-prep<o:p></o:p></span></p>
+  <p class="MsoNormal"><span lang="EN-US" style="mso-bidi-font-weight:bold">backup
+  plates: C1, C2, C3 for part 8<o:p></o:p></span></p>
+  <p class="MsoNormal"><b><span lang="EN-US">Re-streak of <i style="mso-bidi-font-style:
+  normal">E.coli</i> clones from prtas 6 and 7 on new LB<sup>Chloram</sup>
+  plates<o:p></o:p></span></b></p>
+  <p class="MsoNormal"><b><span lang="EN-US">Preparation of new LB<sup>Chloram</sup>
+  plates (500 ml) and Preparation of 250 ml LB broth media (stored on shelf
+  above bench)</span></b><span lang="EN-US" style="mso-bidi-font-weight:bold"><o:p></o:p></span></p>
+  <p class="MsoNormal"><b><span lang="EN-US"><o:p>&nbsp;</o:p></span></b></p>
+  <p class="MsoNormal"><b><span lang="EN-US">PCR with DarR primers with different
+  enzymes and different buffers<o:p></o:p></span></b></p>
+  <p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt;mso-list:
+  l6 level1 lfo2"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:
+  &quot;Times New Roman&quot;;mso-bidi-font-weight:bold"><span style="mso-list:Ignore">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+  </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:
+  bold">dilution of primer stocks 1:20 (100 pmol </span><span lang="EN-US" style="font-family:Wingdings;mso-ascii-font-family:&quot;Times New Roman&quot;;
+  mso-hansi-font-family:&quot;Times New Roman&quot;;mso-char-type:symbol;mso-symbol-font-family:
+  Wingdings;mso-bidi-font-weight:bold"><span style="mso-char-type:symbol;
+  mso-symbol-font-family:Wingdings">à</span></span><span lang="EN-US" style="mso-bidi-font-weight:bold"> 5 pmol):<o:p></o:p></span></p>
+  <p class="MsoNormal" align="center" style="margin-left:36.0pt;text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">95 µl HPLC water + 5 µl primer
+pmol<o:p></o:p></span></p>
+  <p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt;mso-list:
+  l6 level1 lfo2"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:
+  &quot;Times New Roman&quot;;mso-bidi-font-weight:bold"><span style="mso-list:Ignore">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+  </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:
+  bold">preparation of dNTP mix </span><span lang="EN-US" style="font-family:
+  Wingdings;mso-ascii-font-family:&quot;Times New Roman&quot;;mso-hansi-font-family:&quot;Times New Roman&quot;;
+  mso-char-type:symbol;mso-symbol-font-family:Wingdings;mso-bidi-font-weight:
+  bold"><span style="mso-char-type:symbol;mso-symbol-font-family:Wingdings">à</span></span><span lang="EN-US" style="mso-bidi-font-weight:bold"> dilute stocks 1:8 (100 mM </span><span lang="EN-US" style="font-family:Wingdings;mso-ascii-font-family:&quot;Times New Roman&quot;;
+  mso-hansi-font-family:&quot;Times New Roman&quot;;mso-char-type:symbol;mso-symbol-font-family:
+  Wingdings;mso-bidi-font-weight:bold"><span style="mso-char-type:symbol;
+  mso-symbol-font-family:Wingdings">à</span></span><span lang="EN-US" style="mso-bidi-font-weight:bold"> 12.5 mM):<o:p></o:p></span></p>
+  <p class="MsoNormal" align="center" style="margin-left:36.0pt;text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">50 µl of dATP 100 mM<o:p></o:p></span></p>
+  <p class="MsoNormal" align="center" style="margin-left:36.0pt;text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">+ 50 µl of dGTP 100 mM<o:p></o:p></span></p>
+  <p class="MsoNormal" align="center" style="margin-left:36.0pt;text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">+ 50 µl of dTTP 100 mM<o:p></o:p></span></p>
+  <p class="MsoNormal" align="center" style="margin-left:36.0pt;text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">+ 50 µl of dCTP 100 mM<o:p></o:p></span></p>
+  <p class="MsoNormal" align="center" style="margin-left:36.0pt;text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">+ 200 µl dH<sub>2</sub>O<o:p></o:p></span></p>
+  <p class="MsoNormal" style="margin-left:54.0pt;text-indent:-18.0pt;mso-list:
+  l8 level1 lfo3"><!--[if !supportLists]--><span lang="EN-US" style="font-family:
+  Wingdings;mso-fareast-font-family:Wingdings;mso-bidi-font-family:Wingdings;
+  mso-bidi-font-weight:bold"><span style="mso-list:Ignore">è<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:bold">diluted primers and dNTP mix
+  stored in red box at -20 °C<o:p></o:p></span></p>
+  <p class="MsoNormal"><b><span lang="EN-US"><o:p>&nbsp;</o:p></span></b></p>
+  <p class="MsoNormal"><span lang="EN-US" style="mso-bidi-font-weight:bold">1x
+  reaction (50 µl)<o:p></o:p></span></p>
+  <table class="MsoNormalTable" border="1" cellspacing="0" cellpadding="0" style="border-collapse:collapse;border:none;mso-border-alt:solid black .5pt;
+   mso-padding-alt:0cm 3.5pt 0cm 3.5pt;mso-border-insideh:.5pt solid black;
+   mso-border-insidev:.5pt solid black">
+   <tbody><tr style="mso-yfti-irow:0;mso-yfti-firstrow:yes;height:14.2pt">
+    <td width="245" valign="top" style="width:184.1pt;border:solid black 1.0pt;
+    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt;height:14.2pt">
+    <p class="MsoNormal"><b><span lang="EN-US" style="font-size:10.0pt">Component<o:p></o:p></span></b></p>
+    </td>
+    <td width="95" valign="top" style="width:70.9pt;border:solid black 1.0pt;
+    border-left:none;mso-border-left-alt:solid black .5pt;mso-border-alt:solid black .5pt;
+    padding:0cm 3.5pt 0cm 3.5pt;height:14.2pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><b><span lang="EN-US" style="font-size:10.0pt">Volume<o:p></o:p></span></b></p>
+    </td>
+   </tr>
+   <tr style="mso-yfti-irow:1;height:14.2pt">
+    <td width="245" valign="top" style="width:184.1pt;border:solid black 1.0pt;
+    border-top:none;mso-border-top-alt:solid black .5pt;mso-border-alt:solid black .5pt;
+    padding:0cm 3.5pt 0cm 3.5pt;height:14.2pt">
+    <p class="MsoNormal"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:
+    bold">Buffer (5x GC buffer or 5x HF buffer)<o:p></o:p></span></p>
+    </td>
+    <td width="95" valign="top" style="width:70.9pt;border-top:none;border-left:
+    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
+    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt;height:14.2pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:bold">10 µl<o:p></o:p></span></p>
+    </td>
+   </tr>
+   <tr style="mso-yfti-irow:2;height:14.2pt">
+    <td width="245" valign="top" style="width:184.1pt;border:solid black 1.0pt;
+    border-top:none;mso-border-top-alt:solid black .5pt;mso-border-alt:solid black .5pt;
+    padding:0cm 3.5pt 0cm 3.5pt;height:14.2pt">
+    <p class="MsoNormal"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:
+    bold">dNTP mix (12.5 mM each)<o:p></o:p></span></p>
+    </td>
+    <td width="95" valign="top" style="width:70.9pt;border-top:none;border-left:
+    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
+    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt;height:14.2pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:bold">2 µl<o:p></o:p></span></p>
+    </td>
+   </tr>
+   <tr style="mso-yfti-irow:3;height:14.2pt">
+    <td width="245" valign="top" style="width:184.1pt;border:solid black 1.0pt;
+    border-top:none;mso-border-top-alt:solid black .5pt;mso-border-alt:solid black .5pt;
+    padding:0cm 3.5pt 0cm 3.5pt;height:14.2pt">
+    <p class="MsoNormal"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:
+    bold">Primer fwd iGEM_34 (5 pmol)<o:p></o:p></span></p>
+    </td>
+    <td width="95" valign="top" style="width:70.9pt;border-top:none;border-left:
+    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
+    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt;height:14.2pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:bold">2 µl<o:p></o:p></span></p>
+    </td>
+   </tr>
+   <tr style="mso-yfti-irow:4;height:14.2pt">
+    <td width="245" valign="top" style="width:184.1pt;border:solid black 1.0pt;
+    border-top:none;mso-border-top-alt:solid black .5pt;mso-border-alt:solid black .5pt;
+    padding:0cm 3.5pt 0cm 3.5pt;height:14.2pt">
+    <p class="MsoNormal"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:
+    bold">Primer rev iGEM_35 (5 pmol)<o:p></o:p></span></p>
+    </td>
+    <td width="95" valign="top" style="width:70.9pt;border-top:none;border-left:
+    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
+    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt;height:14.2pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:bold">2 µl<o:p></o:p></span></p>
+    </td>
+   </tr>
+   <tr style="mso-yfti-irow:5;height:14.2pt">
+    <td width="245" valign="top" style="width:184.1pt;border:solid black 1.0pt;
+    border-top:none;mso-border-top-alt:solid black .5pt;mso-border-alt:solid black .5pt;
+    padding:0cm 3.5pt 0cm 3.5pt;height:14.2pt">
+    <p class="MsoNormal"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:
+    bold">Chromosomal DNA <i style="mso-bidi-font-style:normal">M. smegmatis</i><o:p></o:p></span></p>
+    </td>
+    <td width="95" valign="top" style="width:70.9pt;border-top:none;border-left:
+    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
+    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt;height:14.2pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:bold">2 µl<o:p></o:p></span></p>
+    </td>
+   </tr>
+   <tr style="mso-yfti-irow:6;height:14.2pt">
+    <td width="245" valign="top" style="width:184.1pt;border:solid black 1.0pt;
+    border-top:none;mso-border-top-alt:solid black .5pt;mso-border-alt:solid black .5pt;
+    padding:0cm 3.5pt 0cm 3.5pt;height:14.2pt">
+    <p class="MsoNormal"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:
+    bold">DNA-Polymerase (Phusion or PhuS) or dH<sub>2</sub>O for water control<o:p></o:p></span></p>
+    </td>
+    <td width="95" valign="top" style="width:70.9pt;border-top:none;border-left:
+    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
+    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt;height:14.2pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:bold">1 µl<o:p></o:p></span></p>
+    </td>
+   </tr>
+   <tr style="mso-yfti-irow:7;mso-yfti-lastrow:yes;height:14.2pt">
+    <td width="245" valign="top" style="width:184.1pt;border:solid black 1.0pt;
+    border-top:none;mso-border-top-alt:solid black .5pt;mso-border-alt:solid black .5pt;
+    padding:0cm 3.5pt 0cm 3.5pt;height:14.2pt">
+    <p class="MsoNormal"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:
+    bold">dH<sub>2</sub>O<o:p></o:p></span></p>
+    </td>
+    <td width="95" valign="top" style="width:70.9pt;border-top:none;border-left:
+    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
+    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt;height:14.2pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:bold">31 µl<o:p></o:p></span></p>
+    </td>
+   </tr>
+  </tbody></table>
+  <p class="MsoNormal"><span lang="EN-US" style="mso-bidi-font-weight:bold"><o:p>&nbsp;</o:p></span></p>
+  <p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt;mso-list:
+  l0 level1 lfo4"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:
+  &quot;Times New Roman&quot;;mso-bidi-font-weight:bold"><span style="mso-list:Ignore">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+  </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:
+  bold">preparation of master mix for 6 reactions containing<o:p></o:p></span></p>
+  <table class="MsoNormalTable" border="1" cellspacing="0" cellpadding="0" style="margin-left:36.0pt;border-collapse:collapse;border:none;mso-border-alt:
+   solid black .5pt;mso-padding-alt:0cm 3.5pt 0cm 3.5pt;mso-border-insideh:
+   .5pt solid black;mso-border-insidev:.5pt solid black">
+   <tbody><tr style="mso-yfti-irow:0;mso-yfti-firstrow:yes">
+    <td width="207" valign="top" style="width:155.2pt;border:solid black 1.0pt;
+    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt">
+    <div style="mso-element:para-border-div;border:none windowtext 1.0pt;
+    mso-border-alt:none windowtext 0cm;padding:1.0pt 4.0pt 1.0pt 4.0pt">
+    <p class="MsoNormal" style="tab-stops:center 207.65pt right 415.3pt;
+    layout-grid-mode:char;border:none;mso-border-alt:none windowtext 0cm;
+    padding:0cm;mso-padding-alt:1.0pt 4.0pt 1.0pt 4.0pt"><b><span lang="EN-US" style="font-size:10.0pt">Component<o:p></o:p></span></b></p>
+    </div>
+    </td>
+    <td width="66" valign="top" style="width:49.6pt;border:solid black 1.0pt;
+    border-left:none;mso-border-left-alt:solid black .5pt;mso-border-alt:solid black .5pt;
+    padding:0cm 3.5pt 0cm 3.5pt">
+    <div style="mso-element:para-border-div;border:none windowtext 1.0pt;
+    mso-border-alt:none windowtext 0cm;padding:1.0pt 4.0pt 1.0pt 4.0pt">
+    <p class="MsoNormal" align="center" style="text-align:center;tab-stops:center 207.65pt right 415.3pt;
+    layout-grid-mode:char;border:none;mso-border-alt:none windowtext 0cm;
+    padding:0cm;mso-padding-alt:1.0pt 4.0pt 1.0pt 4.0pt"><b><span lang="EN-US" style="font-size:10.0pt">Volume<o:p></o:p></span></b></p>
+    </div>
+    </td>
+   </tr>
+   <tr style="mso-yfti-irow:1">
+    <td width="207" valign="top" style="width:155.2pt;border:solid black 1.0pt;
+    border-top:none;mso-border-top-alt:solid black .5pt;mso-border-alt:solid black .5pt;
+    padding:0cm 3.5pt 0cm 3.5pt">
+    <div style="mso-element:para-border-div;border:none windowtext 1.0pt;
+    mso-border-alt:none windowtext 0cm;padding:1.0pt 4.0pt 1.0pt 4.0pt">
+    <p class="MsoNormal" style="tab-stops:center 207.65pt right 415.3pt;
+    layout-grid-mode:char;border:none;mso-border-alt:none windowtext 0cm;
+    padding:0cm;mso-padding-alt:1.0pt 4.0pt 1.0pt 4.0pt"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:bold">dNTP mix (12.5 mM each)<o:p></o:p></span></p>
+    </div>
+    </td>
+    <td width="66" valign="top" style="width:49.6pt;border-top:none;border-left:
+    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
+    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt">
+    <div style="mso-element:para-border-div;border:none windowtext 1.0pt;
+    mso-border-alt:none windowtext 0cm;padding:1.0pt 4.0pt 1.0pt 4.0pt">
+    <p class="MsoNormal" align="center" style="text-align:center;tab-stops:center 207.65pt right 415.3pt;
+    layout-grid-mode:char;border:none;mso-border-alt:none windowtext 0cm;
+    padding:0cm;mso-padding-alt:1.0pt 4.0pt 1.0pt 4.0pt"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:bold">12 µl<o:p></o:p></span></p>
+    </div>
+    </td>
+   </tr>
+   <tr style="mso-yfti-irow:2">
+    <td width="207" valign="top" style="width:155.2pt;border:solid black 1.0pt;
+    border-top:none;mso-border-top-alt:solid black .5pt;mso-border-alt:solid black .5pt;
+    padding:0cm 3.5pt 0cm 3.5pt">
+    <div style="mso-element:para-border-div;border:none windowtext 1.0pt;
+    mso-border-alt:none windowtext 0cm;padding:1.0pt 4.0pt 1.0pt 4.0pt">
+    <p class="MsoNormal" style="tab-stops:center 207.65pt right 415.3pt;
+    layout-grid-mode:char;border:none;mso-border-alt:none windowtext 0cm;
+    padding:0cm;mso-padding-alt:1.0pt 4.0pt 1.0pt 4.0pt"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:bold">Primer fwd iGEM_34 (5
+    pmol)<o:p></o:p></span></p>
+    </div>
+    </td>
+    <td width="66" valign="top" style="width:49.6pt;border-top:none;border-left:
+    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
+    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt">
+    <div style="mso-element:para-border-div;border:none windowtext 1.0pt;
+    mso-border-alt:none windowtext 0cm;padding:1.0pt 4.0pt 1.0pt 4.0pt">
+    <p class="MsoNormal" align="center" style="text-align:center;tab-stops:center 207.65pt right 415.3pt;
+    layout-grid-mode:char;border:none;mso-border-alt:none windowtext 0cm;
+    padding:0cm;mso-padding-alt:1.0pt 4.0pt 1.0pt 4.0pt"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:bold">12 µl<o:p></o:p></span></p>
+    </div>
+    </td>
+   </tr>
+   <tr style="mso-yfti-irow:3">
+    <td width="207" valign="top" style="width:155.2pt;border:solid black 1.0pt;
+    border-top:none;mso-border-top-alt:solid black .5pt;mso-border-alt:solid black .5pt;
+    padding:0cm 3.5pt 0cm 3.5pt">
+    <div style="mso-element:para-border-div;border:none windowtext 1.0pt;
+    mso-border-alt:none windowtext 0cm;padding:1.0pt 4.0pt 1.0pt 4.0pt">
+    <p class="MsoNormal" style="tab-stops:center 207.65pt right 415.3pt;
+    layout-grid-mode:char;border:none;mso-border-alt:none windowtext 0cm;
+    padding:0cm;mso-padding-alt:1.0pt 4.0pt 1.0pt 4.0pt"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:bold">Primer rev iGEM_35 (5
+    pmol)<o:p></o:p></span></p>
+    </div>
+    </td>
+    <td width="66" valign="top" style="width:49.6pt;border-top:none;border-left:
+    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
+    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt">
+    <div style="mso-element:para-border-div;border:none windowtext 1.0pt;
+    mso-border-alt:none windowtext 0cm;padding:1.0pt 4.0pt 1.0pt 4.0pt">
+    <p class="MsoNormal" align="center" style="text-align:center;tab-stops:center 207.65pt right 415.3pt;
+    layout-grid-mode:char;border:none;mso-border-alt:none windowtext 0cm;
+    padding:0cm;mso-padding-alt:1.0pt 4.0pt 1.0pt 4.0pt"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:bold">12 µl<o:p></o:p></span></p>
+    </div>
+    </td>
+   </tr>
+   <tr style="mso-yfti-irow:4;mso-yfti-lastrow:yes">
+    <td width="207" valign="top" style="width:155.2pt;border:solid black 1.0pt;
+    border-top:none;mso-border-top-alt:solid black .5pt;mso-border-alt:solid black .5pt;
+    padding:0cm 3.5pt 0cm 3.5pt">
+    <div style="mso-element:para-border-div;border:none windowtext 1.0pt;
+    mso-border-alt:none windowtext 0cm;padding:1.0pt 4.0pt 1.0pt 4.0pt">
+    <p class="MsoNormal" style="tab-stops:center 207.65pt right 415.3pt;
+    layout-grid-mode:char;border:none;mso-border-alt:none windowtext 0cm;
+    padding:0cm;mso-padding-alt:1.0pt 4.0pt 1.0pt 4.0pt"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:bold">dH<sub>2</sub>O<o:p></o:p></span></p>
+    </div>
+    </td>
+    <td width="66" valign="top" style="width:49.6pt;border-top:none;border-left:
+    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
+    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt">
+    <div style="mso-element:para-border-div;border:none windowtext 1.0pt;
+    mso-border-alt:none windowtext 0cm;padding:1.0pt 4.0pt 1.0pt 4.0pt">
+    <p class="MsoNormal" align="center" style="text-align:center;tab-stops:center 207.65pt right 415.3pt;
+    layout-grid-mode:char;border:none;mso-border-alt:none windowtext 0cm;
+    padding:0cm;mso-padding-alt:1.0pt 4.0pt 1.0pt 4.0pt"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:bold">186 µl<o:p></o:p></span></p>
+    </div>
+    </td>
+   </tr>
+  </tbody></table>
+  <p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt;mso-list:
+  l0 level1 lfo4"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:
+  &quot;Times New Roman&quot;;mso-bidi-font-weight:bold"><span style="mso-list:Ignore">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+  </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:
+  bold">addition of template (chrom. DNA/dH<sub>2</sub>O), polymerase and
+  buffer individually, then addition of 37 µl master mix<b><o:p></o:p></b></span></p>
+  <p class="MsoNormal" style="margin-left:54.0pt;text-indent:-18.0pt;mso-list:
+  l7 level1 lfo5"><!--[if !supportLists]--><span lang="EN-US" style="font-family:
+  Wingdings;mso-fareast-font-family:Wingdings;mso-bidi-font-family:Wingdings;
+  mso-bidi-font-weight:bold"><span style="mso-list:Ignore">è<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><!--[endif]--><b><span lang="EN-US">For tested combinations of buffer and Pol: see table for gel
+  loading<o:p></o:p></span></b></p>
+  <p class="MsoNormal" style="margin-left:36.0pt"><b><span lang="EN-US"><o:p>&nbsp;</o:p></span></b></p>
+  <p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt;mso-list:
+  l0 level1 lfo4"><!--[if !supportLists]--><span lang="DE" style="mso-fareast-font-family:
+  &quot;Times New Roman&quot;;mso-ansi-language:DE;mso-bidi-font-weight:bold"><span style="mso-list:Ignore">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+  </span></span></span><!--[endif]--><span lang="DE" style="mso-ansi-language:DE;
+  mso-bidi-font-weight:bold">PCR protocol (cycler 7; folder “Katrin” &gt;
+  “iGEM” &gt; “DarR seq”)<b><o:p></o:p></b></span></p>
+  <table class="MsoNormalTable" border="1" cellspacing="0" cellpadding="0" style="margin-left:36.0pt;border-collapse:collapse;border:none;mso-border-alt:
+   solid black .5pt;mso-padding-alt:0cm 3.5pt 0cm 3.5pt;mso-border-insideh:
+   .5pt solid black;mso-border-insidev:.5pt solid black">
+   <tbody><tr style="mso-yfti-irow:0;mso-yfti-firstrow:yes">
+    <td width="94" valign="top" style="width:70.15pt;border:solid black 1.0pt;
+    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><b><span lang="EN-US" style="font-size:10.0pt">Step<o:p></o:p></span></b></p>
+    </td>
+    <td width="142" valign="top" style="width:106.3pt;border:solid black 1.0pt;
+    border-left:none;mso-border-left-alt:solid black .5pt;mso-border-alt:solid black .5pt;
+    padding:0cm 3.5pt 0cm 3.5pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><b><span lang="EN-US" style="font-size:10.0pt">Temperature<o:p></o:p></span></b></p>
+    </td>
+    <td width="113" valign="top" style="width:3.0cm;border:solid black 1.0pt;
+    border-left:none;mso-border-left-alt:solid black .5pt;mso-border-alt:solid black .5pt;
+    padding:0cm 3.5pt 0cm 3.5pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><b><span lang="EN-US" style="font-size:10.0pt">Time<o:p></o:p></span></b></p>
+    </td>
+   </tr>
+   <tr style="mso-yfti-irow:1;height:38.3pt">
+    <td width="94" valign="top" style="width:70.15pt;border:solid black 1.0pt;
+    border-top:none;mso-border-top-alt:solid black .5pt;mso-border-alt:solid black .5pt;
+    padding:0cm 3.5pt 0cm 3.5pt;height:38.3pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:bold">Initial denaturation<o:p></o:p></span></p>
+    </td>
+    <td width="142" valign="top" style="width:106.3pt;border-top:none;border-left:
+    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
+    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt;height:38.3pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:bold">98.5 °C<o:p></o:p></span></p>
+    </td>
+    <td width="113" valign="top" style="width:3.0cm;border-top:none;border-left:
+    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
+    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt;height:38.3pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:bold">5 min<o:p></o:p></span></p>
+    </td>
+   </tr>
+   <tr style="mso-yfti-irow:2">
+    <td width="94" valign="top" style="width:70.15pt;border:solid black 1.0pt;
+    border-top:none;mso-border-top-alt:solid black .5pt;mso-border-alt:solid black .5pt;
+    padding:0cm 3.5pt 0cm 3.5pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:bold">Denaturation<o:p></o:p></span></p>
+    </td>
+    <td width="142" valign="top" style="width:106.3pt;border-top:none;border-left:
+    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
+    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:bold">98.5 °C<o:p></o:p></span></p>
+    </td>
+    <td width="113" valign="top" style="width:3.0cm;border-top:none;border-left:
+    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
+    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:bold">30 s<o:p></o:p></span></p>
+    </td>
+   </tr>
+   <tr style="mso-yfti-irow:3">
+    <td width="94" valign="top" style="width:70.15pt;border:solid black 1.0pt;
+    border-top:none;mso-border-top-alt:solid black .5pt;mso-border-alt:solid black .5pt;
+    padding:0cm 3.5pt 0cm 3.5pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:bold">Annealing<o:p></o:p></span></p>
+    </td>
+    <td width="142" valign="top" style="width:106.3pt;border-top:none;border-left:
+    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
+    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:bold">60 °C<o:p></o:p></span></p>
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:9.0pt;mso-bidi-font-weight:bold">(T<sub>A</sub> = T<sub>M</sub>
+    (≈66 °C) – 6 °C)<o:p></o:p></span></p>
+    </td>
+    <td width="113" valign="top" style="width:3.0cm;border-top:none;border-left:
+    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
+    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:bold">35 s<o:p></o:p></span></p>
+    </td>
+   </tr>
+   <tr style="mso-yfti-irow:4">
+    <td width="94" valign="top" style="width:70.15pt;border:solid black 1.0pt;
+    border-top:none;mso-border-top-alt:solid black .5pt;mso-border-alt:solid black .5pt;
+    padding:0cm 3.5pt 0cm 3.5pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:bold">Elongation<o:p></o:p></span></p>
+    </td>
+    <td width="142" valign="top" style="width:106.3pt;border-top:none;border-left:
+    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
+    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:bold">72 °C<o:p></o:p></span></p>
+    </td>
+    <td width="113" valign="top" style="width:3.0cm;border-top:none;border-left:
+    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
+    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:bold">2 min (Phu<o:p></o:p></span></p>
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:bold"><span style="mso-spacerun:yes">&nbsp;</span>needs more time than Phusion!)<o:p></o:p></span></p>
+    </td>
+   </tr>
+   <tr style="mso-yfti-irow:5">
+    <td width="94" valign="top" style="width:70.15pt;border:solid black 1.0pt;
+    border-top:none;mso-border-top-alt:solid black .5pt;mso-border-alt:solid black .5pt;
+    padding:0cm 3.5pt 0cm 3.5pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:bold">Final elongation<o:p></o:p></span></p>
+    </td>
+    <td width="142" valign="top" style="width:106.3pt;border-top:none;border-left:
+    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
+    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:bold">72 °C<o:p></o:p></span></p>
+    </td>
+    <td width="113" valign="top" style="width:3.0cm;border-top:none;border-left:
+    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
+    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:bold">10 min<o:p></o:p></span></p>
+    </td>
+   </tr>
+   <tr style="mso-yfti-irow:6;mso-yfti-lastrow:yes">
+    <td width="94" valign="top" style="width:70.15pt;border:solid black 1.0pt;
+    border-top:none;mso-border-top-alt:solid black .5pt;mso-border-alt:solid black .5pt;
+    padding:0cm 3.5pt 0cm 3.5pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:bold">Hold<o:p></o:p></span></p>
+    </td>
+    <td width="142" valign="top" style="width:106.3pt;border-top:none;border-left:
+    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
+    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:bold">15 °C<o:p></o:p></span></p>
+    </td>
+    <td width="113" valign="top" style="width:3.0cm;border-top:none;border-left:
+    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
+    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:10.0pt;mso-bidi-font-weight:bold">∞<o:p></o:p></span></p>
+    </td>
+   </tr>
+  </tbody></table>
+  <p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt;mso-list:
+  l0 level1 lfo4"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:
+  &quot;Times New Roman&quot;;mso-bidi-font-weight:bold"><span style="mso-list:Ignore">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+  </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:
+  bold">1 % Agarose-1xTAE gel<o:p></o:p></span></p>
+  <p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt;mso-list:
+  l0 level1 lfo4"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:
+  &quot;Times New Roman&quot;;mso-bidi-font-weight:bold"><span style="mso-list:Ignore">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+  </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:
+  bold">4 µl PCR reaction + 1 µl 5x DNA Loading Dye<o:p></o:p></span></p>
+  <p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt;mso-list:
+  l0 level1 lfo4"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:
+  &quot;Times New Roman&quot;;mso-bidi-font-weight:bold"><span style="mso-list:Ignore">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+  </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:
+  bold">3 µl 1 kb ladder (Quick Load)<o:p></o:p></span></p>
+  <p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt;mso-list:
+  l0 level1 lfo4"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:
+  &quot;Times New Roman&quot;;mso-bidi-font-weight:bold"><span style="mso-list:Ignore">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+  </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:
+  bold">Run at 100 V in 1xTAE buffer<o:p></o:p></span></p>
+  <p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt;mso-list:
+  l0 level1 lfo4"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:
+  &quot;Times New Roman&quot;;mso-bidi-font-weight:bold"><span style="mso-list:Ignore">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+  </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:
+  bold">Staining in EtBr and destaining in water<o:p></o:p></span></p>
+  <p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt;mso-list:
+  l0 level1 lfo4"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:
+  &quot;Times New Roman&quot;;mso-bidi-font-weight:bold"><span style="mso-list:Ignore">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+  </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:
+  bold">UV detection<o:p></o:p></span></p>
+  <table class="MsoNormalTable" border="1" cellspacing="0" cellpadding="0" style="border-collapse:collapse;border:none;mso-border-alt:solid black .5pt;
+   mso-padding-alt:0cm 3.5pt 0cm 3.5pt;mso-border-insideh:.5pt solid black;
+   mso-border-insidev:.5pt solid black">
+   <tbody><tr style="mso-yfti-irow:0;mso-yfti-firstrow:yes">
+    <td width="57" valign="top" style="width:42.7pt;border:solid black 1.0pt;
+    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">M<o:p></o:p></span></p>
+    </td>
+    <td width="57" valign="top" style="width:42.7pt;border:solid black 1.0pt;
+    border-left:none;mso-border-left-alt:solid black .5pt;mso-border-alt:solid black .5pt;
+    padding:0cm 3.5pt 0cm 3.5pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">1<o:p></o:p></span></p>
+    </td>
+    <td width="57" valign="top" style="width:42.7pt;border:solid black 1.0pt;
+    border-left:none;mso-border-left-alt:solid black .5pt;mso-border-alt:solid black .5pt;
+    padding:0cm 3.5pt 0cm 3.5pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">2<o:p></o:p></span></p>
+    </td>
+    <td width="57" valign="top" style="width:42.7pt;border:solid black 1.0pt;
+    border-left:none;mso-border-left-alt:solid black .5pt;mso-border-alt:solid black .5pt;
+    padding:0cm 3.5pt 0cm 3.5pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">3<o:p></o:p></span></p>
+    </td>
+    <td width="57" valign="top" style="width:42.7pt;border:solid black 1.0pt;
+    border-left:none;mso-border-left-alt:solid black .5pt;mso-border-alt:solid black .5pt;
+    padding:0cm 3.5pt 0cm 3.5pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">4<o:p></o:p></span></p>
+    </td>
+    <td width="57" valign="top" style="width:42.7pt;border:solid black 1.0pt;
+    border-left:none;mso-border-left-alt:solid black .5pt;mso-border-alt:solid black .5pt;
+    padding:0cm 3.5pt 0cm 3.5pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">5<o:p></o:p></span></p>
+    </td>
+    <td width="57" valign="top" style="width:42.75pt;border:solid black 1.0pt;
+    border-left:none;mso-border-left-alt:solid black .5pt;mso-border-alt:solid black .5pt;
+    padding:0cm 3.5pt 0cm 3.5pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">M<o:p></o:p></span></p>
+    </td>
+   </tr>
+   <tr style="mso-yfti-irow:1">
+    <td width="57" valign="top" style="width:42.7pt;border:solid black 1.0pt;
+    border-top:none;mso-border-top-alt:solid black .5pt;mso-border-alt:solid black .5pt;
+    padding:0cm 3.5pt 0cm 3.5pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">1 kb ladder<o:p></o:p></span></p>
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">(QuickLoad)<o:p></o:p></span></p>
+    </td>
+    <td width="57" valign="top" style="width:42.7pt;border-top:none;border-left:
+    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
+    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">HF<o:p></o:p></span></p>
+    </td>
+    <td width="57" valign="top" style="width:42.7pt;border-top:none;border-left:
+    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
+    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">GC<o:p></o:p></span></p>
+    </td>
+    <td width="57" valign="top" style="width:42.7pt;border-top:none;border-left:
+    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
+    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">HF<o:p></o:p></span></p>
+    </td>
+    <td width="57" valign="top" style="width:42.7pt;border-top:none;border-left:
+    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
+    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">GC<o:p></o:p></span></p>
+    </td>
+    <td width="57" valign="top" style="width:42.7pt;border-top:none;border-left:
+    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
+    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">Water controlHF<o:p></o:p></span></p>
+    </td>
+    <td width="57" valign="top" style="width:42.75pt;border-top:none;border-left:
+    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
+    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">1 kb ladder<o:p></o:p></span></p>
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">(QuickLoad)<o:p></o:p></span></p>
+    </td>
+   </tr>
+   <tr style="mso-yfti-irow:2;mso-yfti-lastrow:yes">
+    <td width="57" valign="top" style="width:42.7pt;border:solid black 1.0pt;
+    border-top:none;mso-border-top-alt:solid black .5pt;mso-border-alt:solid black .5pt;
+    padding:0cm 3.5pt 0cm 3.5pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold"><o:p>&nbsp;</o:p></span></p>
+    </td>
+    <td width="114" colspan="2" valign="top" style="width:85.4pt;border-top:none;
+    border-left:none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
+    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">Phusion<o:p></o:p></span></p>
+    </td>
+    <td width="171" colspan="3" valign="top" style="width:128.1pt;border-top:none;
+    border-left:none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
+    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">PhuS<o:p></o:p></span></p>
+    </td>
+    <td width="57" valign="top" style="width:42.75pt;border-top:none;border-left:
+    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+    mso-border-top-alt:solid black .5pt;mso-border-left-alt:solid black .5pt;
+    mso-border-alt:solid black .5pt;padding:0cm 3.5pt 0cm 3.5pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold"><o:p>&nbsp;</o:p></span></p>
+    </td>
+   </tr>
+  </tbody></table>
+  <p class="MsoNormal"><b><span lang="EN-US"><!--[if gte vml 1]><v:shapetype id="_x0000_t75"
+   coordsize="21600,21600" o:spt="75" o:preferrelative="t" path="m@4@5l@4@11@9@11@9@5xe"
+   filled="f" stroked="f">
+   <v:stroke joinstyle="miter"/>
+   <v:formulas>
+    <v:f eqn="if lineDrawn pixelLineWidth 0"/>
+    <v:f eqn="sum @0 1 0"/>
+    <v:f eqn="sum 0 0 @1"/>
+    <v:f eqn="prod @2 1 2"/>
+    <v:f eqn="prod @3 21600 pixelWidth"/>
+    <v:f eqn="prod @3 21600 pixelHeight"/>
+    <v:f eqn="sum @0 0 1"/>
+    <v:f eqn="prod @6 1 2"/>
+    <v:f eqn="prod @7 21600 pixelWidth"/>
+    <v:f eqn="sum @8 21600 0"/>
+    <v:f eqn="prod @7 21600 pixelHeight"/>
+    <v:f eqn="sum @10 21600 0"/>
+   </v:formulas>
+   <v:path o:extrusionok="f" gradientshapeok="t" o:connecttype="rect"/>
+   <o:lock v:ext="edit" aspectratio="t"/>
+  </v:shapetype><v:shape id="_x0000_i1025" type="#_x0000_t75" style='width:300pt;
+   height:223.5pt;mso-position-horizontal-relative:page;
+   mso-position-vertical-relative:page'>
+   <v:imagedata src="part1.files/image001.png" o:title="DarR different enzymes"/>
+  </v:shape><![endif]--><!--[if !vml]--><img src="https://static.igem.org/mediawiki/2013/3/30/Goe-10.6.13-RT.png" v:shapes="_x0000_i1025"><!--[endif]--><o:p></o:p></span></b></p>
+  <p class="MsoNormal" style="margin-left:54.0pt;text-indent:-18.0pt;mso-list:
+  l7 level1 lfo5"><!--[if !supportLists]--><span lang="EN-US" style="font-family:
+  Wingdings;mso-fareast-font-family:Wingdings;mso-bidi-font-family:Wingdings;
+  mso-bidi-font-weight:bold"><span style="mso-list:Ignore">è<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:bold">Reactions stored at - 20°C in 50
+  ml Falcon<b><o:p></o:p></b></span></p>
-<b>Plasmid Mini-Preparation of parts 8, 7 Cm</b>
+<div class="fbutton">Fold ↑</div>
-<ul><li>harvesting of ca. 5 ml pellets in 2 ml Epis for future Plasmid Mini-Preparation ? stored at – 20 °C in red box</li>
-<li>harvesting of remaining culture for today’s prep</li>
-<li>Performed as on 7.June.13</li></ul>
-<p>NanoDrop – Plasmid concentrations</p>
-<table cellspacing="0">
-<tr><th>Part Number</th><th>c(DNA)[ng/μl]</th><th>A<sub>260</sub>/A<sub>280</sub></th><th>A<sub>260</sub>/A<sub>230</sub></th></tr>
-<tr><td>7</td><td>67.0</td><td>1.98</td><td>2.25</td></tr>
-<tr><td>8</td><td>62.7</td><td>1.89</td><td>2.08</td></tr>
-</table>
-<b>DarR seq PCR purification</b>
-<ul>
-<li>for DarR seq PCR reactions 1 - 4</li>
-<li>with QIAquick PCR purification Kit (Qiagen), according to quick start manual</li>
-<li>ca. 50 μl of PCR reaction + 250 μl of PB buffer</li>
-<li>reactions 2 and 4 turned violet ? addition of 10 μl NaAc 3.3 M, pH = 5.0</li>
-<li>elution: 30 μl pre-warmed HPLC water directly applied to membrane, incubation for 2 min at RT, centrifugation</li>
-<li>purified PCR products stored at – 20 °C in red box</li>
-</ul>
 </div>
 </div>
 </div>
-<div class="tlob" id="tl_0608">
-<span class="date">08th<img src="https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png" /></span>
-<div class="cont">
-<p class="timeline-title">Cloning DarR ORF from g-DNA</p>
-<div class="timeline-cont">
-<b>Colonies on the plates: parts 8, 7 C1</b>
-<p>10 ml LB medium with 10 μl antibiotics, overnight culture for mini-prep</p>
-<p>backup plates: C1, C2, C3 for part 8</p>
-<b>Colonies on the plates: parts 8, 7 C1</b>
-<p>Preparation of 250 ml LB broth media (stored on shelf above bench)</p>
-<p>10 ml LB medium with 10 μl antibiotics, overnight culture for mini-prep</p>
-<p>backup plates: C1, C2, C3 for part 8</p>
-<b>Re-streak of E.coli clones from prtas 6 and 7 on new LBChloram plates</b>
-<b>Preparation of new LBChloram plates (500 ml) and Preparation of 250 ml LB broth media (stored on shelf above bench)</b>
-<b>PCR with DarR primers with different enzymes and different buffers</b>
-<ul>
-<li>dilution of primer stocks 1:20 (100 pmol -> 5 pmol):95 μl HPLC water + 5 μl primer 100 pmol</li>
-<li>preparation of dNTP mix -> dilute stocks 1:8 (100 mM ? 12.5 mM):50μl dNTP + 200μl dH<sub>2</sub>O</li>
-<li>diluted primers and dNTP mix stored in red box at -20 °C</li>
-</ul>
-<p>1x reaction(50μl)</p>
-<table cellspacing="0">
-<tr><th>Component</th><th>Volume(μl)</th></tr>
-<tr><td>Buffer (5x GC buffer or 5x HF buffer)</td><td>10</td></tr>
-<tr><td>dNTP mix (12.5 mM each)</td><td>2</td></tr>
-<tr><td>Primer fwd iGEM_34 (5 pmol)</td><td>2</td></tr>
-<tr><td>Primer rev iGEM_35 (5 pmol)</td><td>2</td></tr>
-<tr><td>Chromosomal DNA M. smegmatis</td><td>2</td></tr>
-<tr><td>DNA-Polymerase (Phusion or PhuS)<br /> or dH2O for water control</td><td>1</td></tr>
-<tr><td>dH2O</td><td>31</td></tr>
-</table>
-<p>Master Mix for 6 reactions</p>
-<table>
-<tr><th>Component</th><th>Volume(μl)</th></tr>
-<tr><td>dNTP mix (12.5 mM each)</td><td>12</td></tr>
-<tr><td>Primer fwd iGEM_34 (5 pmol)</td><td>12</td></tr>
-<tr><td>Primer rev iGEM_35 (5 pmol)</td><td>12</td></tr>
-<tr><td>dH2O</td><td>186</td></tr>
-<table>
-<p>addition of template (chrom. DNA/dH2O), polymerase and buffer individually, then addition of 37 μl master mix</p>
-<p><b>For tested combinations of buffer and Pol: see table for gel loading</b></p>
-<p>PCR protocol (cycler 7; folder “Katrin” > “iGEM” > “DarR seq”)</p>
-<table>
-<tr><th>Step</th><th>Temperature</th><th>Time</th></tr>
-<tr><td>Initial denaturation</td><td>98.5 °C</td><td>5 min</td></tr>
-<tr><td>Denaturation</td><td>98.5 °C</td><td>30 s</td></tr>
-<tr><td>Annealing</td><td>60 °C<br />(TA = TM (≈66 °C) – 6 °C)
-</td><td>35 s</td></tr>
-<tr><td>Elongation</td><td>72 °C</td><td>2 min (Phu
- needs more time than Phusion!)</td></tr>
-<tr><td>Final elongation</td><td>72 °C</td><td>10 min</td></tr>
-<tr><td>Hold</td><td>15 °C</td><td>∞</td></tr>
-</table>
-<ul>
-<li>Protocol of 1 % Agarose-1xTAE gel</li>
-<li>4 μl PCR reaction + 1 μl 5x DNA Loading Dye</li>
-<li>3 μl 1 kb ladder (Quick Load)</li>
-<li>Run at 100 V in 1xTAE buffer</li>
-<li>Staining in EtBr and destaining in water</li>
-<li>UV detection</li>
-</ul>
-<p>Loading scheme</p>
-<table>
-<tr><th>M</th><th>1</th><th>2</th><th>3</th><th>4</th><th>5</th><th>M</th></tr>
-<tr><td>1kb ladder<br />(QuickLoad)</td><td>HF</td><td>GC</td><td>HF</td><td>GC</td><td>Water control <br />with HF</td><td>1 kb ladder<br />(QuickLoad)</td>
-</table>
-<img src="https://static.igem.org/mediawiki/2013/3/30/Goe-10.6.13-RT.png" />
-<p>Reactions stored at - 20°C in 50 ml Falcon</p>
-</div>
-</div>
-</div>
 </html>