Team:Goettingen/NoteBook w3

From 2013.igem.org

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Revision as of 19:52, 30 September 2013

21st<img src="" />

Transformation from yesterday worked!

Transformation from yesterday worked! 6 clones! And the Without-Insert-Control lacks clones!

è Plates stored at 4 °C<o:p></o:p>

20th<img src="">

Digestion of PCR product (DarR Operator) (10 cycles) and Plasmid backbone (pSB1A3)

Concentration of stored PCR products from <st1:chsdate isrocdate="False" islunardate="False" day="30" month="12" year="1899" w:st="on">19.6.13</st1:chsdate><o:p></o:p>

No. of PCR cycles<o:p></o:p>	c(DNA) [ng/µl]	A₂₆₀/A₂₈₀	A₂₆₀/A₂₃₀
PCR product<o:p></o:p> 10 cycles<o:p></o:p>	1068.7	1.63	1.07
PCR product<o:p></o:p> 20 cycles<o:p></o:p>	975.7	1.66	1.10
PCR product<o:p></o:p> 30 cycles<o:p></o:p>	964.9	1.65	1.11

<o:p> </o:p>

Digestion of PCR product (AND WHATS IN THE PCR?) (10 cycles) and Plasmid backbone (pSB1A3 (with AmpR))<o:p></o:p>

According to: <a href="http://parts.igem.org/Help:Protocols/Linearized_Plasmid_Backbones">http://parts.igem.org/Help:Protocols/Linearized_Plasmid_Backbones</a>

<o:p> </o:p>

Ezyme master mix:

5 µl Buffer 0<o:p></o:p>

0.5 µl EcoRI<o:p></o:p>

0.5 µl PstI<o:p></o:p>

18 µl dH₂O<o:p></o:p>

<o:p> </o:p>

Digestion of backbone resp. insert:<o:p></o:p>

4 µl linearized plasmid backbone (25 ng/µl for 100 ng total)<o:p></o:p>

4 µl Enzyme master mix<o:p></o:p>

resp.:<o:p></o:p>

5 µl insert<o:p></o:p>

4 µl Enzyme master mix<o:p></o:p>

resp.:<o:p></o:p>

10 µl insert<o:p></o:p>

4 µl Enzyme master mix<o:p></o:p>

<o:p> </o:p>

digest 37°C/30 min heat kill 80°C/20 min<o:p></o:p>

<o:p> </o:p>

Ligation of backbone and insert:<o:p></o:p>

According to: <a href="http://parts.igem.org/Help:Protocols/Linearized_Plasmid_Backbones">http://parts.igem.org/Help:Protocols/Linearized_Plasmid_Backbones</a>

<o:p> </o:p>

2 µl digested plasmid backbone<o:p></o:p>

10 µl EcoRI and PstI digested fragment <o:p></o:p>

1.5 µl T4 DNA Ligase buffer<o:p></o:p>

0.5 µl T4 DNA Ligase<o:p></o:p>

1 µl H₂O<o:p></o:p>

ligate 30°C/30 min heat kill 80°C/min<o:p></o:p>

<o:p> </o:p>

Transformation of ligated parts <o:p></o:p>

According to method book<o:p></o:p>

Digestion of amplified DAC-domains (S.aureus and S. pneumonia) from 17.06.

Digestion of amplified DAC-domains (S.aureus and S. pneumonia) from 17.06.

Components:	Volume
plasmid DNA	16µl
PstI	6µl
EcoRI	6µl
orange buffer (10x)	8µl
H₂O	44µl

è incubation for 30min at 37°C

è heat inactivation for 20min at 65°C

è store at -20°C

Digestion of prepared Strep-Tag from 17.06.

Components:	Volume
Strep-Tag	16µl
PstI	6µl
EcoRI	6µl
orange buffer (10x)	8µl
H₂O	44µl

è incubation for 30min at 37°C

è heat inactivation for 20min at 65°C

è store at -20°C

è Preparation of Strap-Tag worked

è Preparation of DAC domain of S.aureus worked

è Preparation of DAC domain of S- pneumonia failed = EcoRI restriction site in fragment

Fold ↑

19th<img src="">

PCR using primer iGEM 36,37 for construction of DarR operator biobrick,DarR sequencing

PCR using primer iGEM 36,37 for construction of DarR operator biobrick.<o:p></o:p>

Same recipe as yesterdays PCR, rest the protocol,

98.5	5min	<o:p> </o:p>	<o:p> </o:p>	<o:p> </o:p>
98.5	30s	10 cycle	20cycle	30cycle
61.0	30s
72.0	2min
72.0	10min	<o:p> </o:p>	<o:p> </o:p>	<o:p> </o:p>

Ctrl: no enzyme<o:p></o:p>

Increase the gel concentration to 4%.

PCR worked, Gel here:

Lane 1-8: ladder, iGEM36, iGEM37, ctrl(no Polyermase), 1 cycle, 10cycle, 20cycle, 30cycle.

Gel concentration : 4%

1cycle PCR product from yesterday.

So, use 4% gel, 10cycle PCR is enough<o:p></o:p>

DarR sequencing results<o:p></o:p>

in “our” genomic DNA of M. smegmatis, there are two additional codons inserted right after the GTG start codon:

“our” gDNA: GTG AGC GCG AGC GCC à Ser-Ala-Ser-Ala

Paper gDNA: GTG _ _ _ _ _ _ AGC GCC à Ser-Ala

Additionally, there are 5 point mutations:

120 C à G

132 A à G

210 T à C

228 C à T

263 T à C

All are silent, except for one: aa 87 Val à Ala (both small, hydrophobic aa)

Primer Design<o:p></o:p>

Design of new DarR biobrick fwd primer (since 2 additional codons inserted and old primer designed for DarR gene without these codons)<o:p></o:p>

18th<img src="" />

Test restriction of Part 2 C2,Primer design,Construction of DarR operator biobrick(PCR)

Plate reader assay<o:p></o:p>

- Plate thrown away after ON measurement<o:p></o:p>

- Results:<o:p></o:p>

<o:p> </o:p>

Concentration of plasmid purified from Clone 2 of Part 2<o:p></o:p>

Part no.<o:p></o:p>

c(DNA)<o:p></o:p>

[ng/µl]<o:p></o:p>

A₂₆₀/A₂₈₀<o:p></o:p>

A₂₆₀/A₂₃₀<o:p></o:p>

2 C2<o:p></o:p>

120.6<o:p></o:p>

1.87<o:p></o:p>

2.24<o:p></o:p>

<o:p> </o:p>

Test restriction of Part 2 Clone 2<o:p></o:p>

Component<o:p></o:p>	Uncut control<o:p></o:p>	SpeI single digestion<o:p></o:p>	PstI single digestion<o:p></o:p>	SpeI/PstI double digestion<o:p></o:p>
Plasmid DNA (150 ng)<o:p></o:p>	1.2 µl<o:p></o:p>	1.2 µl<o:p></o:p>	1.2 µl<o:p></o:p>	1.2 µl<o:p></o:p>
10x buffer<o:p></o:p>	1 µl (Tango)<o:p></o:p>	1 µl (Tango)<o:p></o:p>	1 µl (buffer 0)<o:p></o:p>	1 µl (Tango)<o:p></o:p>
SpeI<o:p></o:p>	-<o:p></o:p>	1 µl<o:p></o:p>	-<o:p></o:p>	1 µl<o:p></o:p>
PstI<o:p></o:p>	-<o:p></o:p>	-<o:p></o:p>	1 µl<o:p></o:p>	2 µl<o:p></o:p>
dH₂O<o:p></o:p>	7.8 µl<o:p></o:p>	6.8 µl<o:p></o:p>	6.8 µl<o:p></o:p>	4.8 µl<o:p></o:p>
Total<o:p></o:p>	10 µl<o:p></o:p>	10 µl<o:p></o:p>	10 µl<o:p></o:p>	10 µl<o:p></o:p>

- Incubation for c<st1:chsdate year="1899" month="12" day="30" islunardate="False" isrocdate="False" w:st="on">a. 1.5</st1:chsdate> h at 37 °C (incubator)<o:p></o:p>

- Addition of 2 µl of 5x DNA loading dye and run on 1 % 1xTAE-agarose gel (marker: 100 bp ladder)<o:p></o:p>

- EtBr staining and destaining in H₂O, UV detection<o:p></o:p>

è Gel: See ppt-file “dropbox > iGEM > Reporter-Team > Geldoc“<o:p></o:p>

<img src="" width="350px" v:shapes="_x0000_i1027"><o:p></o:p>

Conclusions:<o:p></o:p>

è Clone 2 contains the expected plasmid harboring part 2 (impure stock?)<o:p></o:p>

<o:p> </o:p>

Primer design and ordering<o:p></o:p>

Design of primer iGEM_43<o:p></o:p>

Ordering of primers iGEM_38 to iGEM_43 from Sigma-Aldrich<o:p></o:p>

<o:p> </o:p>

PCR:<o:p></o:p>

PCR using primer iGEM32,33 for construction of DarR biobrick.

Because of those two primers have high tendency to form 2^nd structure, 1x 2x 4x primers are applied, to see the recipe, see dropbox > Reporter Team > PCR_iGEM32_33.xls

PCR was successful, better use 2x primer. Protocol as follows:<o:p></o:p>

98.5	5min	<o:p> </o:p>
98.5	30s	30 cycles
67.0	35s
72.0	2min
72.0	10min	<o:p> </o:p>

<o:p> </o:p>

è Gel: See ppt-file “dropbox > iGEM > Reporter-Team > Geldoc“<o:p></o:p>

<img src="" width="350px" v:shapes="Objekt_x0020_3"><o:p></o:p>

PCR using primer iGEM 36,37 for construction of DarR operator biobrick. To see the recipe, see dropbox > Reporter Team > PCR_iGEM32_33.xls

Protocol as follows:

98.5	5min	Only one cycle
98.5	30s
61.0	30s
72.0	12min

<o:p> </o:p>

è Gel: See ppt-file “dropbox > iGEM > Reporter-Team > Geldoc“<o:p></o:p>

<o:p> </o:p>

2% gel cannot separate the primers and PCR product:

iGEM36:36nt

iGEM37:35nt

PCR product should be 57bp

Gel1: forget the primer as ctrl

Gel2: load the wrong primer

<o:p> </o:p>

 </td>

17th<img src="" />

Plate reader assay for the pink strains, Plasmid mini prep for Part C2

Cryo-Stocks of E. coli transfomant (2, C2)<o:p></o:p>

Stored in -70 °C (red box)<o:p></o:p>

<o:p> </o:p>

Plasmid Mini-Preparation of part 2 C2<o:p></o:p>

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

- harvesting of remaining culture for today’s prep<o:p></o:p>

è Performed as on <st1:chsdate isrocdate="False" islunardate="False" day="30" month="12" year="1899" w:st="on">7.6.13</st1:chsdate><o:p></o:p>

<o:p> </o:p>

Plate reader assay<o:p></o:p>

- Measurment of OD of ON cultures in 1:10 dilution in LB; LB as blank (see table)<o:p></o:p>

- Preparation of E.coli cultures of an OD_600nm = 0.05 in LB (without antibiotics) in 96-well microtiterplate; <o:p></o:p>

final volume: 180 µl (see table) à triplicates for each clone<o:p></o:p>

Calculation:<o:p></o:p>

V(well culture) * OD_600nm (wanted)/ OD_600nm (ON-culture) = V(ON culture needed for preparation of well cultures)<o:p></o:p>

180 µl * 0.05/OD_600nm (ON-culture) = V(ON culture needed for preparation of well cultures) [µ]<o:p></o:p>

<o:p> </o:p>

Values and amounts for well cultures:<o:p></o:p>

Clone<o:p></o:p>		OD600nm <o:p></o:p> (ON culture)<o:p></o:p> 1:10<o:p></o:p>	OD600nm<o:p></o:p> (ON culture)<o:p></o:p> 1:1<o:p></o:p>	V(ON culture for preparation of well culture)[µ]<o:p></o:p>
DHα<o:p></o:p>		0.433<o:p></o:p>	4.33<o:p></o:p>	2.1<o:p></o:p>
Part 1<o:p></o:p>	C1<o:p></o:p>	0.426<o:p></o:p>	4.26<o:p></o:p>	2.1<o:p></o:p>
Part 1<o:p></o:p>	C2<o:p></o:p>	0.450<o:p></o:p>	4.50<o:p></o:p>	2<o:p></o:p>
Part 2<o:p></o:p>	C1<o:p></o:p>	0.416<o:p></o:p>	4.16<o:p></o:p>	2.2<o:p></o:p>
	C2<o:p></o:p>	0.436<o:p></o:p>	4.36<o:p></o:p>	2.1<o:p></o:p>
	C3<o:p></o:p>	0.434<o:p></o:p>	4.34<o:p></o:p>	2.1<o:p></o:p>
Part 3<o:p></o:p>	C1<o:p></o:p>	0.467<o:p></o:p>	4.67<o:p></o:p>	1.9<o:p></o:p>
	C2<o:p></o:p>	0.475<o:p></o:p>	4.75<o:p></o:p>	1.9<o:p></o:p>
	C3<o:p></o:p>	0.473<o:p></o:p>	4.73<o:p></o:p>	1.9<o:p></o:p>
Part 4<o:p></o:p>	C1<o:p></o:p>	0.457<o:p></o:p>	4.57<o:p></o:p>	2<o:p></o:p>
	C 2<o:p></o:p>	0.466<o:p></o:p>	4.66<o:p></o:p>	1.9<o:p></o:p>
	C3<o:p></o:p>	0.437<o:p></o:p>	4.37<o:p></o:p>	2.1<o:p></o:p>
Part 6<o:p></o:p>	C1<o:p></o:p>	0.450<o:p></o:p>	4.50<o:p></o:p>	2<o:p></o:p>
	C2<o:p></o:p>	0.431<o:p></o:p>	4.31<o:p></o:p>	2.1<o:p></o:p>
	C3<o:p></o:p>	0.448<o:p></o:p>	4.48<o:p></o:p>	2<o:p></o:p>

è Pipetting of ON-culture volume into each well, then addition of 180 µl LB without antibiotics using multi-channel pipett (be careful: tips must be fixed properly!)<o:p></o:p>

è For scheme: see folder<o:p></o:p>

- Incubation of plate at 37 °C with vortexing in plate reader, fluorescence of GFP and RFP, as well as OD_600nm measured every 15 min for approx. 3 h<o:p></o:p>

<o:p> </o:p>

Fluorescence excited and measured at:<o:p></o:p>

<o:p> </o:p>	Absorption<o:p></o:p>	Emission<o:p></o:p>
RFP<o:p></o:p>	555 nm<o:p></o:p>	583 nm<o:p></o:p>
GFP<o:p></o:p>	489 nm<o:p></o:p>	509 nm<o:p></o:p>

- Storage of plate at 4 °C<o:p></o:p>

- Measurement ON under same conditions<o:p></o:p>

Preperation of Strep-Tag for Biobrick assembly

Preperation of Strep-Tag for Biobrick assembly

Components:	Volume (µl)
Primer iGEM_11 (5µM)	5µl
Primer iGEM_12 (5µM)	5µl
dNTPs	1µl
buffer (5x)	10µl
H₂O	28µl

è incubation at 90°C for 5min, cool down at room temperature and add 1µl of PfuS polymerase

è incubation at 72°C for 15min

Amplification of S. aureus and S. pneumonia DAC domains for Biobrick assembly

Components:	Volume
Primer iGEM_13 (5µM)	2µl
Primer iGEM_14 (5µM)	2µl
Primer iGEM_15 (5µM)	2µl
Primer iGEM_16 (5µM)	2µl
genomic DNA	2µl
dNTPs	2µl
buffer (5x)	10µl
H₂O	33µl
PfuS polymerase	1µl

Fold ↑

16th<img src="" />

Innoculation of Part 3 C2

@@ Line 1: / Line 1: @@
-<html>
-<style>
-.timeline-cont{display:none}
-</style>
 <div class="tlob" id="tl_0621">
 <span class="date">21st<img src="https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png" /></span>
 <div class="cont">
-<p class="timeline-title">Transformation
+<p class="timeline-title goe-rt">Transformation
    from yesterday worked!</p>
 <div class="timeline-cont">
@@ Line 20: / Line 15: @@
-<div class="fbutton">Fold ↑</div>
 </div>
 </div>
@@ Line 29: / Line 23: @@
 <span class="date">20th<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">Digestion of PCR product (DarR Operator) (10 cycles) and Plasmid backbone (pSB1A3)</p>
+<p class="timeline-title goe-rt" title="Click to fold/extend">Digestion of PCR product (DarR Operator) (10 cycles) and Plasmid backbone (pSB1A3)</p>
-<div class="timeline-cont">
+<div class="timeline-cont" style="display: block;">
    <p class="MsoNormal"><b style="mso-bidi-font-weight:normal"><span lang="EN-US">Concentration
@@ Line 183: / Line 177: @@
+</div>
+<p class="timeline-title goe-dac">Digestion of amplified DAC-domains (S.aureus and S.
+  pneumonia) from 17.06.</p>
+<div class="timeline-cont">
+  <p class="MsoNormal"><b><span lang="EN-US" style="font-size:11.0pt;font-family:
+  &quot;Arial&quot;,&quot;sans-serif&quot;">Digestion of amplified DAC-domains (S.aureus and S.
+  pneumonia) from 17.06.</span></b></p>
+  <p class="MsoNormal"><b><span lang="EN-US" style="font-size:11.0pt;font-family:
+  &quot;Arial&quot;,&quot;sans-serif&quot;">&nbsp;</span></b></p>
+  <table class="MsoTableGrid" border="1" cellspacing="0" cellpadding="0" style="border-collapse:collapse;border:none">
+   <tbody><tr>
+    <td width="292" valign="top" style="width:219.1pt;border:solid black 1.0pt;
+    padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">Components:</span></p>
+    </td>
+    <td width="236" valign="top" style="width:176.9pt;border:solid black 1.0pt;
+    border-left:none;padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">Volume</span></p>
+    </td>
+   </tr>
+   <tr>
+    <td width="292" valign="top" style="width:219.1pt;border:solid black 1.0pt;
+    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoListParagraphCxSpFirst" style="margin-bottom:0cm;margin-bottom:
+    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">&nbsp;</span></p>
+    <p class="MsoListParagraphCxSpMiddle" style="margin-bottom:0cm;margin-bottom:
+    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">plasmid
+    DNA</span></p>
+    </td>
+    <td width="236" valign="top" style="width:176.9pt;border-top:none;border-left:
+    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+    padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoListParagraphCxSpMiddle" style="margin-bottom:0cm;margin-bottom:
+    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">&nbsp;</span></p>
+    <p class="MsoListParagraphCxSpLast" style="margin-bottom:0cm;margin-bottom:
+    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">16µl</span></p>
+    </td>
+   </tr>
+   <tr>
+    <td width="292" valign="top" style="width:219.1pt;border:solid black 1.0pt;
+    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoListParagraphCxSpFirst" style="margin-bottom:0cm;margin-bottom:
+    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">PstI</span></p>
+    </td>
+    <td width="236" valign="top" style="width:176.9pt;border-top:none;border-left:
+    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+    padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoListParagraphCxSpLast" style="margin-bottom:0cm;margin-bottom:
+    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">6µl</span></p>
+    </td>
+   </tr>
+   <tr>
+    <td width="292" valign="top" style="width:219.1pt;border:solid black 1.0pt;
+    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoListParagraphCxSpFirst" style="margin-bottom:0cm;margin-bottom:
+    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">EcoRI</span></p>
+    </td>
+    <td width="236" valign="top" style="width:176.9pt;border-top:none;border-left:
+    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+    padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoListParagraphCxSpLast" style="margin-bottom:0cm;margin-bottom:
+    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">6µl</span></p>
+    </td>
+   </tr>
+   <tr>
+    <td width="292" valign="top" style="width:219.1pt;border:solid black 1.0pt;
+    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoListParagraphCxSpFirst" style="margin-bottom:0cm;margin-bottom:
+    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">orange
+    buffer (10x)</span></p>
+    </td>
+    <td width="236" valign="top" style="width:176.9pt;border-top:none;border-left:
+    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+    padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoListParagraphCxSpLast" style="margin-bottom:0cm;margin-bottom:
+    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">8µl</span></p>
+    </td>
+   </tr>
+   <tr>
+    <td width="292" valign="top" style="width:219.1pt;border:solid black 1.0pt;
+    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoListParagraphCxSpFirst" style="margin-bottom:0cm;margin-bottom:
+    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">H<sub>2</sub>O</span></p>
+    </td>
+    <td width="236" valign="top" style="width:176.9pt;border-top:none;border-left:
+    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+    padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoListParagraphCxSpLast" style="margin-bottom:0cm;margin-bottom:
+    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">44µl</span></p>
+    </td>
+   </tr>
+  </tbody></table>
+  <p class="MsoListParagraphCxSpFirst" style="margin-top:0cm;margin-right:0cm;
+  margin-bottom:0cm;margin-left:54.0pt;margin-bottom:.0001pt;text-indent:-18.0pt;
+  line-height:normal"><span lang="EN-US" style="font-family:Wingdings">è<span style="font:7.0pt &quot;Times New Roman&quot;"> </span></span><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">incubation for 30min at 37°C</span></p>
+  <p class="MsoListParagraphCxSpMiddle" style="margin-top:0cm;margin-right:0cm;
+  margin-bottom:0cm;margin-left:54.0pt;margin-bottom:.0001pt;text-indent:-18.0pt;
+  line-height:normal"><span lang="EN-US" style="font-family:Wingdings">è<span style="font:7.0pt &quot;Times New Roman&quot;"> </span></span><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">heat inactivation for 20min at 65°C</span></p>
+  <p class="MsoListParagraphCxSpMiddle" style="margin-top:0cm;margin-right:0cm;
+  margin-bottom:0cm;margin-left:54.0pt;margin-bottom:.0001pt;text-indent:-18.0pt;
+  line-height:normal"><span lang="EN-US" style="font-family:Wingdings">è<span style="font:7.0pt &quot;Times New Roman&quot;"> </span></span><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">store at -20°C</span></p>
+  <p class="MsoListParagraphCxSpLast" style="margin-top:0cm;margin-right:0cm;
+  margin-bottom:0cm;margin-left:54.0pt;margin-bottom:.0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">&nbsp;</span></p>
+  <p class="MsoNormal"><b><span lang="EN-US" style="font-size:11.0pt;font-family:
+  &quot;Arial&quot;,&quot;sans-serif&quot;">Digestion of prepared Strep-Tag from 17.06.</span></b></p>
+  <p class="MsoNormal"><b><span lang="EN-US" style="font-size:11.0pt;font-family:
+  &quot;Arial&quot;,&quot;sans-serif&quot;">&nbsp;</span></b></p>
+  <table class="MsoTableGrid" border="1" cellspacing="0" cellpadding="0" style="border-collapse:collapse;border:none">
+   <tbody><tr>
+    <td width="292" valign="top" style="width:219.1pt;border:solid black 1.0pt;
+    padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">Components:</span></p>
+    </td>
+    <td width="236" valign="top" style="width:176.9pt;border:solid black 1.0pt;
+    border-left:none;padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">Volume</span></p>
+    </td>
+   </tr>
+   <tr>
+    <td width="292" valign="top" style="width:219.1pt;border:solid black 1.0pt;
+    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoListParagraphCxSpFirst" style="margin-bottom:0cm;margin-bottom:
+    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">Strep-Tag</span></p>
+    </td>
+    <td width="236" valign="top" style="width:176.9pt;border-top:none;border-left:
+    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+    padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoListParagraphCxSpLast" style="margin-bottom:0cm;margin-bottom:
+    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">16µl</span></p>
+    </td>
+   </tr>
+   <tr>
+    <td width="292" valign="top" style="width:219.1pt;border:solid black 1.0pt;
+    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoListParagraphCxSpFirst" style="margin-bottom:0cm;margin-bottom:
+    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">PstI</span></p>
+    </td>
+    <td width="236" valign="top" style="width:176.9pt;border-top:none;border-left:
+    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+    padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoListParagraphCxSpLast" style="margin-bottom:0cm;margin-bottom:
+    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">6µl</span></p>
+    </td>
+   </tr>
+   <tr>
+    <td width="292" valign="top" style="width:219.1pt;border:solid black 1.0pt;
+    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoListParagraphCxSpFirst" style="margin-bottom:0cm;margin-bottom:
+    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">EcoRI</span></p>
+    </td>
+    <td width="236" valign="top" style="width:176.9pt;border-top:none;border-left:
+    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+    padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoListParagraphCxSpLast" style="margin-bottom:0cm;margin-bottom:
+    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">6µl</span></p>
+    </td>
+   </tr>
+   <tr>
+    <td width="292" valign="top" style="width:219.1pt;border:solid black 1.0pt;
+    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoListParagraphCxSpFirst" style="margin-bottom:0cm;margin-bottom:
+    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">orange
+    buffer (10x)</span></p>
+    </td>
+    <td width="236" valign="top" style="width:176.9pt;border-top:none;border-left:
+    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+    padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoListParagraphCxSpLast" style="margin-bottom:0cm;margin-bottom:
+    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">8µl</span></p>
+    </td>
+   </tr>
+   <tr>
+    <td width="292" valign="top" style="width:219.1pt;border:solid black 1.0pt;
+    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoListParagraphCxSpFirst" style="margin-bottom:0cm;margin-bottom:
+    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">H<sub>2</sub>O</span></p>
+    </td>
+    <td width="236" valign="top" style="width:176.9pt;border-top:none;border-left:
+    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+    padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoListParagraphCxSpLast" style="margin-bottom:0cm;margin-bottom:
+    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">44µl</span></p>
+    </td>
+   </tr>
+  </tbody></table>
+  <p class="MsoListParagraphCxSpFirst" style="margin-bottom:0cm;margin-bottom:
+  .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">&nbsp;</span></p>
+  <p class="MsoListParagraphCxSpMiddle" style="margin-top:0cm;margin-right:0cm;
+  margin-bottom:0cm;margin-left:54.0pt;margin-bottom:.0001pt;text-indent:-18.0pt;
+  line-height:normal"><span lang="EN-US" style="font-family:Wingdings">è<span style="font:7.0pt &quot;Times New Roman&quot;"> </span></span><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">incubation for 30min at 37°C</span></p>
+  <p class="MsoListParagraphCxSpMiddle" style="margin-top:0cm;margin-right:0cm;
+  margin-bottom:0cm;margin-left:54.0pt;margin-bottom:.0001pt;text-indent:-18.0pt;
+  line-height:normal"><span lang="EN-US" style="font-family:Wingdings">è<span style="font:7.0pt &quot;Times New Roman&quot;"> </span></span><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">heat inactivation for 20min at 65°C</span></p>
+  <p class="MsoListParagraphCxSpMiddle" style="margin-top:0cm;margin-right:0cm;
+  margin-bottom:0cm;margin-left:54.0pt;margin-bottom:.0001pt;text-indent:-18.0pt;
+  line-height:normal"><span lang="EN-US" style="font-family:Wingdings">è<span style="font:7.0pt &quot;Times New Roman&quot;"> </span></span><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">store at -20°C</span></p>
+  <p class="MsoListParagraphCxSpMiddle" style="margin-top:0cm;margin-right:0cm;
+  margin-bottom:0cm;margin-left:54.0pt;margin-bottom:.0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">&nbsp;</span></p>
+  <p class="MsoListParagraphCxSpMiddle" style="margin-top:0cm;margin-right:0cm;
+  margin-bottom:0cm;margin-left:54.0pt;margin-bottom:.0001pt;text-indent:-18.0pt;
+  line-height:normal"><span lang="EN-US" style="font-family:Wingdings;color:#00B050">è<span style="font:7.0pt &quot;Times New Roman&quot;"> </span></span><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;;color:#00B050">Preparation of
+  Strap-Tag worked</span></p>
+  <p class="MsoListParagraphCxSpMiddle" style="margin-top:0cm;margin-right:0cm;
+  margin-bottom:0cm;margin-left:54.0pt;margin-bottom:.0001pt;text-indent:-18.0pt;
+  line-height:normal"><span lang="EN-US" style="font-family:Wingdings;color:#00B050">è<span style="font:7.0pt &quot;Times New Roman&quot;"> </span></span><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;;color:#00B050">Preparation of DAC
+  domain of S.aureus worked</span></p>
+  <p class="MsoListParagraphCxSpLast" style="margin-top:0cm;margin-right:0cm;
+  margin-bottom:0cm;margin-left:54.0pt;margin-bottom:.0001pt;text-indent:-18.0pt;
+  line-height:normal"><span lang="EN-US" style="font-family:Wingdings;color:#00B050">è<span style="font:7.0pt &quot;Times New Roman&quot;"> </span></span><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;;color:#00B050">Preparation of DAC
+  domain of S- pneumonia failed = EcoRI restriction site in fragment</span></p>
+  <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">&nbsp;</span></p>
 <div class="fbutton">Fold ↑</div>
 </div>
@@ Line 193: / Line 400: @@
 <span class="date">19th<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">PCR using primer iGEM 36,37 for construction of DarR operator biobrick,DarR sequencing</p>
+<p class="timeline-title goe-rt" title="Click to fold/extend">PCR using primer iGEM 36,37 for construction of DarR operator biobrick,DarR sequencing</p>
-<div class="timeline-cont">
+<div class="timeline-cont" style="display: block;">
    <p class="MsoNormal"><b><span lang="EN-US">PCR using primer iGEM 36<span class="GramE">,37</span> for construction of DarR operator biobrick.<o:p></o:p></span></b></p>
    <p class="MsoNormal"><span lang="EN-US">Same recipe as yesterdays PCR, rest the
@@ Line 376: / Line 583: @@
    without these codons)<b style="mso-bidi-font-weight:normal"><o:p></o:p></b></span></p>
-<div class="fbutton">Fold ↑</div>
 </div>
 </div>
@@ Line 386: / Line 592: @@
 <span class="date">18th<img src="https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png" /></span>
 <div class="cont">
-<p class="timeline-title">Test restriction of Part 2 C2,Primer design,Construction of DarR operator biobrick(PCR)</p>
+<p class="timeline-title goe-rt">Test restriction of Part 2 C2,Primer design,Construction of DarR operator biobrick(PCR)</p>
 <div class="timeline-cont">
@@ Line 929: / Line 1,135: @@
    bold"><o:p>&nbsp;</o:p></span></p>
    </td>
-<div class="fbutton">Fold ↑</div>
 </div>
 </div>
@@ Line 938: / Line 1,142: @@
 <span class="date">17th<img src="https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png" /></span>
 <div class="cont">
-<p class="timeline-title">Plate reader assay for the pink strains, Plasmid mini prep for Part C2</p>
+<p class="timeline-title goe-rt">Plate reader assay for the pink strains, Plasmid mini prep for Part C2</p>
 <div class="timeline-cont">
@@ Line 1,451: / Line 1,655: @@
    l5 level1 lfo12"><!--[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>
+   </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:
-  <img src="https://static.igem.org/mediawiki/2013/6/61/Goe-17.06.13-RT.png" width="500" />
-  <!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:
    bold">Incubation of plate at 37 °C with vortexing in plate reader,
    fluorescence of GFP and RFP, as well as OD<sub>600nm</sub> measured every 15
@@ Line 1,531: / Line 1,733: @@
    </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:
    bold">Measurement ON under same conditions<b><o:p></o:p></b></span></p>
+</div>
+<p class="timeline-title goe-dac">Preperation of Strep-Tag for Biobrick assembly</p>
+<div class="timeline-cont">
+  <p class="MsoNormal"><b><span lang="EN-US" style="font-size:11.0pt;font-family:
+  &quot;Arial&quot;,&quot;sans-serif&quot;">Preperation of Strep-Tag for Biobrick assembly</span></b></p>
+  <p class="MsoNormal"><b><span lang="EN-US" style="font-size:11.0pt;font-family:
+  &quot;Arial&quot;,&quot;sans-serif&quot;">&nbsp;</span></b></p>
+  <table class="MsoTableGrid" border="1" cellspacing="0" cellpadding="0" style="border-collapse:collapse;border:none">
+   <tbody><tr>
+    <td width="292" valign="top" style="width:219.1pt;border:solid black 1.0pt;
+    padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">Components:</span></p>
+    </td>
+    <td width="236" valign="top" style="width:176.9pt;border:solid black 1.0pt;
+    border-left:none;padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">Volume (µl)</span></p>
+    </td>
+   </tr>
+   <tr>
+    <td width="292" valign="top" style="width:219.1pt;border:solid black 1.0pt;
+    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoListParagraphCxSpFirst" style="margin-bottom:0cm;margin-bottom:
+    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">Primer
+    iGEM_11 (5µM)</span></p>
+    </td>
+    <td width="236" valign="top" style="width:176.9pt;border-top:none;border-left:
+    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+    padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoListParagraphCxSpLast" style="margin-bottom:0cm;margin-bottom:
+    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">5µl</span></p>
+    </td>
+   </tr>
+   <tr>
+    <td width="292" valign="top" style="width:219.1pt;border:solid black 1.0pt;
+    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoListParagraphCxSpFirst" style="margin-bottom:0cm;margin-bottom:
+    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">Primer
+    iGEM_12 (5µM)</span></p>
+    </td>
+    <td width="236" valign="top" style="width:176.9pt;border-top:none;border-left:
+    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+    padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoListParagraphCxSpLast" style="margin-bottom:0cm;margin-bottom:
+    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">5µl</span></p>
+    </td>
+   </tr>
+   <tr>
+    <td width="292" valign="top" style="width:219.1pt;border:solid black 1.0pt;
+    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoListParagraphCxSpFirst" style="margin-bottom:0cm;margin-bottom:
+    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">dNTPs</span></p>
+    </td>
+    <td width="236" valign="top" style="width:176.9pt;border-top:none;border-left:
+    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+    padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoListParagraphCxSpLast" style="margin-bottom:0cm;margin-bottom:
+    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">1µl</span></p>
+    </td>
+   </tr>
+   <tr>
+    <td width="292" valign="top" style="width:219.1pt;border:solid black 1.0pt;
+    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoListParagraphCxSpFirst" style="margin-bottom:0cm;margin-bottom:
+    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">buffer
+    (5x)</span></p>
+    </td>
+    <td width="236" valign="top" style="width:176.9pt;border-top:none;border-left:
+    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+    padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoListParagraphCxSpLast" style="margin-bottom:0cm;margin-bottom:
+    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">10µl</span></p>
+    </td>
+   </tr>
+   <tr>
+    <td width="292" valign="top" style="width:219.1pt;border:solid black 1.0pt;
+    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoListParagraphCxSpFirst" style="margin-bottom:0cm;margin-bottom:
+    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">H<sub>2</sub>O</span></p>
+    </td>
+    <td width="236" valign="top" style="width:176.9pt;border-top:none;border-left:
+    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+    padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoListParagraphCxSpLast" style="margin-bottom:0cm;margin-bottom:
+    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">28µl</span></p>
+    </td>
+   </tr>
+  </tbody></table>
+  <p class="MsoListParagraphCxSpFirst" style="margin-bottom:0cm;margin-bottom:
+  .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">&nbsp;</span></p>
+  <p class="MsoListParagraphCxSpMiddle" style="margin-left:54.0pt;text-indent:
+  -18.0pt;line-height:normal"><span lang="EN-US" style="font-family:Wingdings">è<span style="font:7.0pt &quot;Times New Roman&quot;"> </span></span><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">incubation at 90°C for 5min, cool
+  down at room temperature and add 1µl of PfuS polymerase</span></p>
+  <p class="MsoListParagraphCxSpLast" style="margin-left:54.0pt;text-indent:-18.0pt;
+  line-height:normal"><span lang="EN-US" style="font-family:Wingdings">è<span style="font:7.0pt &quot;Times New Roman&quot;"> </span></span><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">incubation at 72°C for 15min</span></p>
+  <p class="MsoNormal"><span lang="EN-US" style="font-size:14.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">&nbsp;</span></p>
+  <p class="MsoNormal"><b><span lang="EN-US" style="font-size:11.0pt;font-family:
+  &quot;Arial&quot;,&quot;sans-serif&quot;">Amplification of S. aureus and S. pneumonia DAC domains
+  for Biobrick assembly</span></b></p>
+  <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">&nbsp;</span></p>
+  <table class="MsoTableGrid" border="1" cellspacing="0" cellpadding="0" style="border-collapse:collapse;border:none">
+   <tbody><tr>
+    <td width="292" valign="top" style="width:219.25pt;border:solid black 1.0pt;
+    padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">Components:</span></p>
+    </td>
+    <td width="236" valign="top" style="width:176.75pt;border:solid black 1.0pt;
+    border-left:none;padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">Volume</span></p>
+    </td>
+   </tr>
+   <tr>
+    <td width="292" valign="top" style="width:219.25pt;border:solid black 1.0pt;
+    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoListParagraphCxSpFirst" style="margin-bottom:0cm;margin-bottom:
+    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">Primer
+    iGEM_13 (5µM)</span></p>
+    </td>
+    <td width="236" valign="top" style="width:176.75pt;border-top:none;border-left:
+    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+    padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoListParagraphCxSpLast" style="margin-bottom:0cm;margin-bottom:
+    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">2µl</span></p>
+    </td>
+   </tr>
+   <tr>
+    <td width="292" valign="top" style="width:219.25pt;border:solid black 1.0pt;
+    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoListParagraphCxSpFirst" style="margin-bottom:0cm;margin-bottom:
+    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">Primer
+    iGEM_14 (5µM)</span></p>
+    </td>
+    <td width="236" valign="top" style="width:176.75pt;border-top:none;border-left:
+    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+    padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoListParagraphCxSpLast" style="margin-bottom:0cm;margin-bottom:
+    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">2µl</span></p>
+    </td>
+   </tr>
+   <tr>
+    <td width="292" valign="top" style="width:219.25pt;border:solid black 1.0pt;
+    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoListParagraphCxSpFirst" style="margin-bottom:0cm;margin-bottom:
+    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">Primer
+    iGEM_15 (5µM)</span></p>
+    </td>
+    <td width="236" valign="top" style="width:176.75pt;border-top:none;border-left:
+    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+    padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoListParagraphCxSpLast" style="margin-bottom:0cm;margin-bottom:
+    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">2µl</span></p>
+    </td>
+   </tr>
+   <tr>
+    <td width="292" valign="top" style="width:219.25pt;border:solid black 1.0pt;
+    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoListParagraphCxSpFirst" style="margin-bottom:0cm;margin-bottom:
+    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">Primer
+    iGEM_16 (5µM)</span></p>
+    </td>
+    <td width="236" valign="top" style="width:176.75pt;border-top:none;border-left:
+    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+    padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoListParagraphCxSpLast" style="margin-bottom:0cm;margin-bottom:
+    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">2µl</span></p>
+    </td>
+   </tr>
+   <tr>
+    <td width="292" valign="top" style="width:219.25pt;border:solid black 1.0pt;
+    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoListParagraphCxSpFirst" style="margin-bottom:0cm;margin-bottom:
+    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">genomic
+    DNA</span></p>
+    </td>
+    <td width="236" valign="top" style="width:176.75pt;border-top:none;border-left:
+    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+    padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoListParagraphCxSpLast" style="margin-bottom:0cm;margin-bottom:
+    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">2µl</span></p>
+    </td>
+   </tr>
+   <tr>
+    <td width="292" valign="top" style="width:219.25pt;border:solid black 1.0pt;
+    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoListParagraphCxSpFirst" style="margin-bottom:0cm;margin-bottom:
+    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">dNTPs</span></p>
+    </td>
+    <td width="236" valign="top" style="width:176.75pt;border-top:none;border-left:
+    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+    padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoListParagraphCxSpLast" style="margin-bottom:0cm;margin-bottom:
+    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">2µl</span></p>
+    </td>
+   </tr>
+   <tr>
+    <td width="292" valign="top" style="width:219.25pt;border:solid black 1.0pt;
+    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoListParagraphCxSpFirst" style="margin-bottom:0cm;margin-bottom:
+    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">buffer
+    (5x)</span></p>
+    </td>
+    <td width="236" valign="top" style="width:176.75pt;border-top:none;border-left:
+    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+    padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoListParagraphCxSpLast" style="margin-bottom:0cm;margin-bottom:
+    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">10µl</span></p>
+    </td>
+   </tr>
+   <tr>
+    <td width="292" valign="top" style="width:219.25pt;border:solid black 1.0pt;
+    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoListParagraphCxSpFirst" style="margin-bottom:0cm;margin-bottom:
+    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">H<sub>2</sub>O</span></p>
+    </td>
+    <td width="236" valign="top" style="width:176.75pt;border-top:none;border-left:
+    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+    padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoListParagraphCxSpLast" style="margin-bottom:0cm;margin-bottom:
+    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">33µl</span></p>
+    </td>
+   </tr>
+   <tr>
+    <td width="292" valign="top" style="width:219.25pt;border:solid black 1.0pt;
+    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoListParagraphCxSpFirst" style="margin-bottom:0cm;margin-bottom:
+    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">PfuS
+    polymerase</span></p>
+    </td>
+    <td width="236" valign="top" style="width:176.75pt;border-top:none;border-left:
+    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+    padding:0cm 5.4pt 0cm 5.4pt">
+    <p class="MsoListParagraphCxSpLast" style="margin-bottom:0cm;margin-bottom:
+    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">1µl</span></p>
+    </td>
+   </tr>
+  </tbody></table>
+  <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">&nbsp;</span></p>
@@ Line 1,542: / Line 1,983: @@
 <span class="date">16th<img src="https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png" /></span>
 <div class="cont">
-<p class="timeline-title">Innoculation of Part 3 C2</p>
+<p class="timeline-title goe-rt">Innoculation of Part 3 C2</p>
+<div class="timeline-cont">
+</div>
 </div>
 </div>
-</html>