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Latest revision as of 11:14, 27 October 2013

Notebook: June

03.06.2013

Digest

Investigator: Franzi

Aim: Construction of P_NR-test-vector → Digestion of combined 2 BioBricks.

10 µl DNA template
0,5 µl enzyme 1
0,5 µl enzyme 2
2 µl Cut Smart buffer
7 µl bidest. H2O

Samples:

Digestion of pSB1A3 iBB1+5 with XbaI and PstI-HF

Digestion of pSB1A3 iBB6+3 with EcoRI-HF and SpeI-HF.

The samples were incubated for 1 h at 37 °C.

They were purified via QIAgen Gel Extraction Kit (Qiagen). Elution into 20 µl bidest. H2O.

Ligation

Investigator: Franzi

Aim: Construction of P_NR-test-vector → Ligation of two combined 2 BioBricks in pSB1C3.

45 ng vector DNA (pSB1C3; EcoRI/PstI-cut)
140 ng iBB6+3 (EcoRI/PstI-cut)
150 ng iBB1+5 (EcoRI/PstI-cut)
2 µl 10x T4 DNA ligase buffer
1 µl T4 DNA ligase
ad 20 µl bidest. H2O

The samples were incubated for 1 h at RT.

Transformation

Investigator: Franzi

Aim: Construction of P_NR-test-vector → Transformation of combined 4 BioBricks in pSB1C3 into E. coli DH5α.

Complete ligation samples were transformed into E. coli DH5α (30 min on ice, 60 sec 42 °C, 60 min 37 °C + 900 µl LB), plated on LB_CM, overnight at 37 °C.

Ligation

Investigator: Franzi

Aim: Construction of BioBricks → Ligation of single BioBricks into pSB1C3.

45 ng vector DNA (pSB1C3; EcoRI/PstI-cut)
X ng insert
1 µl 10x T4 DNA ligase buffer
1 µl T4 DNA ligase
ad 10 µl bidest. H2O

Samples (all EcoRI/PstI-cut):

120 ng eGFP (iBB9)

60 ng SPTP1 (iBB10)

50 ng SPTP2 (iBB11)

55 ng SP1 (iBB12)

50 ng SP2 (iBB13)

The samples were incubated for 3 h at RT.

Transformation

Investigator: Franzi

Aim: Construction of BioBricks → Transformation of single BioBricks in pSB1C3 into E. coli DH5α.

Complete ligation samples were transformed into E. coli DH5α (30 min on ice, 60 sec 42 °C, 60 min 37 °C + 900 µl LB), plated on LB_CM, overnight at 37 °C.

Inoculation

Investigator: Franzi

Aim: Construction of P_fcpB-test-vector and antibody-vector → Inoculation of transformants for miniprep.

3 colonies of pSB1A3 iBB1+6 inoculated in 5 ml LB_amp, 4 colonies of pSB1C3 iBB4864 inoculated in 5 ml LB_CM, overnight at 37 °C.

04.06.2013

Miniprep

Investigator: Franzi

Aim: Construction of P_fcpB-test-vector and antibody-vector → Miniprep of pSB1A3 iBB1+6 and pSB1C3 iBB4864.

Miniprep of the liquid cultures (“QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf)), eluted in 30 µl bidest. H2O. 2 Cultures of pSB1C3 iBB4864 discarded (red colour).

Digest

Investigator: Franzi

Aim: Construction of P_fcpB-test-vector and antibody-vector → Control digestion of pSB1A3 iBB1+6 and pSB1C3 iBB4864.

1 µl DNA template
0,5 µl EcoRI-HF
0,5 µl PstI-HF
1 µl Cut Smart buffer
7 µl bidest. H2O

The samples were incubated for 2 h at 37 °C.

Gel electrophoresis

Gel substances

1% Agarose gel
10 µl RedSafe per 50 ml gel
6 µl 2-Log DNA Ladder (NEB)
6x loading buffer (for samples)

Lane	Content	Expectations
2+3	iBB4864 K1-2	2580 bp
4	empty
5-7	iBB16 K3-5	650 bp

Digest

Investigator: Franzi

Aim: Construction of P_fcpB-test-vector and antibody-vector → Digestion of combined 2 BioBricks in pSB1A3 and combined 4 BioBricks in pSB1C3.

10 µl DNA template
0,5 µl enzyme 1
0,5 µl enzyme 2
2 µl Cut Smart buffer
7 µl bidest. H2O

Samples:

Digestion of pSB1C3 iBB7631 K4 and pSB1A3 iBB16 K4 with XbaI and PstI-HF

Digestion of pSB1C3 iBB4864 K1 and pSB1A3 iBB54 K2 with EcoRI-HF and SpeI-HF.

The samples were incubated for 1 h at 37 °C.

They were purified via QIAgen Gel Extraction Kit (Qiagen). Elution into 20 µl bidest. H2O.

Ligation

Investigator: Franzi

Aim: Construction of P_fcpB-test-vector and antibody-vector → Ligation of two combined 2 BioBricks into pSB1C3 and two combined 4 BioBricks into pSB1A3.

Antibody:

80 ng vector DNA (pSB1A3; EcoRI/PstI-cut)
160 ng iBB7631
190 ng iBB4864
1 µl 10x T4 DNA ligase buffer
1 µl T4 DNA ligase
ad 10 µl bidest. H2O

Test-vector:

45 ng vector DNA (pSB1C3; EcoRI/PstI-cut)
150 ng iBB54
120 ng iBB16
1 µl 10x T4 DNA ligase buffer
1 µl T4 DNA ligase
ad 10 µl bidest. H2O

Both samples were incubated overnight at 16 °C.

Inoculation

Investigator: Franzi

Aim: Construction of P_NR-test-vector and BioBricks → Inoculation of transformants.

Transformations (03.06.13) inoculated in 5 ml LB_CM, overnight at 37 °C.

05.06.2013

Miniprep

Investigator: Franzi

Aim: Construction of P_NR-test-vector and BioBricks → Miniprep of single BioBricks and pSB1C3 iBB6315.

Miniprep of the liquid cultures (“QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf)), eluted in 30 µl bidest. H2O.

Digest

Investigator: Franzi

Aim: Construction of P_NR-test-vector and BioBricks → Control digestion of single BioBricks and pSB1C3 iBB6315.

1 µl DNA template
0,3 µl EcoRI-HF
0,3 µl PstI-HF
1 µl Cut Smart buffer
7,4 µl bidest. H2O

The samples were incubated for 1,5 h at 37 °C.

Gel electrophoresis

Gel substances

1% Agarose gel
10 µl RedSafe per 50 ml gel
6 µl 2-Log DNA Ladder (NEB)
6x loading buffer (for samples)

Lane	Content	Expectations
2-4	iBB9 K1-3	780 bp
5	empty
6-8	iBB10 K1-3	228 bp
		pSB1C3: 2 kb

Lane	Content	Expectations
2-4	iBB11 K1-3	147 bp
5	empty
6-8	iBB12 K1-3	123 bp
		pSB1C3: 2 kb

Lane	Content	Expectations
2-4	iBB13 K1-3	108 bp
5	empty
6-8	iBB6315 K1-3	1200 bp
		pSB1C3: 2 kb

Transformation

Investigator: Franzi

Aim: Construction of P_fcpB-test-vector and antibody-vector → Transformation of combined 4 BioBricks in pSB1C3 and combined 8 BioBricks in pSB1A3 into E. coli DH5α.

Ligations (04.06.13) were transformed into E. coli DH5α (30 min on ice, 60 sec 42 °C, 60 min (1C3)/40 min (1A3) 37 °C + 900 µl LB), plated on LB_CM (1C3)/LB_amp (1A3), overnight at 37 °C.

Sequencing

Investigator: Franzi

Aim: Construction of BioBricks → Examination of sequencing results of various constructs.

iBB9 K1 fw - AGB000K 263

iBB9 K1 rv - AGB000K 264

iBB9 K2 fw - AGB000K 265

iBB9 K2 rv - AGB000K 266

iBB10 K1 fw - AGB000K 267

iBB10 K2 fw - AGB000K 268

iBB11 K1 fw - AGB000K 269

iBB11 K2 fw - AGB000K 270

iBB12 K1 fw - AGB000K 271

iBB12 K2 fw - AGB000K 272

iBB13 K1 fw - AGB000K 273

iBB13 K2 fw - AGB000K 274

all correct, except iBB10 G→T pos.30, iBB11 T→C pos.24. Probably template mutated.

06.06.2013

Inoculation

Investigator: Franzi

Aim: Construction of P_fcpB-test-vector and antibody-vector → Inoculation of pSB1C3 iBB5416 and pSB1A3 iBB48647631 for miniprep.

3 colonies of pSB1C3 iBB5416 inoculated in 5 ml LB_CM, 4 colonies of pSB1A3 iBB48647631 inoculated in 5 ml LB_amp, overnight at 37 °C.

07.06.2013

Miniprep

Investigator: Patrick

Aim: Construction of P_fcpB-test-vector and antibody-vector → Miniprep of pSB1C3 iBB5416 and pSB1A3 iBB48647631.

Miniprep of the liquid cultures (“QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf)), eluted in 30 µl bidest. H2O.

Digest

Investigator: Patrick

Aim: Construction of P_fcpB-test-vector and antibody-vector → Control digestion of pSB1C3 iBB5416 and pSB1A3 iBB48647631.

1 µl DNA template
0,3 µl EcoRI-HF
0,3 µl PstI-HF
1 µl Cut Smart buffer
7,4 µl bidest. H2O

The samples were incubated for 1 h at 37 °C.

Gel electrophoresis

Gel substances

1% Agarose gel
10 µl RedSafe per 50 ml gel
6 µl 2-Log DNA Ladder (NEB)
6x loading buffer (for samples)

Lane

Content

Expectations

2-4

iBB5416 K1-3

pSB1C3: 2070 bp

iBB5416: 1500 bp

5-8

iBB48647631 K1-4

pSB1A3: 2150 bp

iBB48647631: 4157 bp

10.06.2013

Digest

Investigator: Lukas

Aim: Construction of P_NR-test-vector, P_fcpB-test-vector and antibody-vector → Digestion of pSB1A3 iBB48647631 and single BioBricks.

X µl DNA template
0,5 µl enzyme 1
0,5 µl enzyme 2
2,2 µl Cut Smart buffer
20 µl bidest. H2O

Samples:

Digestion of 1,5 µl pSB1A3 iBB48647631 with SpeI-HF and PstI-HF

Digestion of 5 µl pSB1C3 iBB5 and iBB9 with XbaI and PstI-HF

Digestion of 4 µl pSB1C3 iBB3 and iBB4 with EcoRI-HF and SpeI-HF.

The samples were incubated for 1 h at 37 °C.

They were purified via QIAgen Gel Extraction Kit (Qiagen). Elution into 20 µl bidest. H2O.

Ligation

Investigator: Christian

Aim: Construction of P_NR-test-vector, P_fcpB-test-vector and antibody-vector → Ligation of pSB1A3 iBB48647631 and single BioBricks in pSB1A3.

Ligation of pSB1A3 iBB48647631 with iBB5, pSB1A3 iBB48647631 with iBB3 and iBB9, pSB1A3 iBB48647631 with iBB4 and iBB9; overnight at 16°C

11.06.2013

Transformation

Investigator: Franzi

Aim: Construction of P_NR-test-vector, P_fcpB-test-vector and antibody-vector %rarr; Transformation of ligations.

Transformation of the ligations into E. coli DH5α (30 min on ice, 60 sec 42 °C, 25 min 37 °C + 900 µl LB), plated on LB_amp, overnight at 37 °C.

12.06.2013

Inoculation

Investigator: Franzi

Aim: Construction of P_NR-test-vector, P_fcpB-test-vector and antibody-vector → Inoculation of transformants for miniprep.

4 colonies of each transformation inoculated in 4 ml LB_amp, overnight at 37 °C.

13.06.2013

Miniprep

Investigator: Franzi

Aim: Construction of P_NR-test-vector, P_fcpB-test-vector and antibody-vector → Miniprep of transformants.

Miniprep of the liquid cultures (“QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf)), eluted in 30 µl bidest. H2O.

Digest

Investigator: Franzi

Aim: Construction of P_NR-test-vector, P_fcpB-test-vector and antibody-vector → Control digestion the minipreps and pSB1A3 iBB48647631 K3 (comparison of size).

1 µl DNA template
0,3 µl EcoRI-HF
0,3 µl PstI-HF
1 µl Cut Smart buffer
7,4 µl bidest. H2O

The samples were incubated for 1 h at 37 °C.

Gel electrophoresis

Gel substances

1% Agarose gel
10 µl RedSafe per 50 ml gel
6 µl 2-Log DNA Ladder (NEB)
6x loading buffer (for samples)

Lane	Content	Expectations
upper:
2+3	iBB49 K3-4	1260 bp
4-7	iBB39 K1-4	1030 bp
lower:
2	iBB49647631	4157 bp
3-6	iBB496476315 K1-4	4404 bp
7+8	iBB49 K1-2	1260 bp
		Vector pSB1A3: 2150 bp

IBB48647631 and iBB486476315 probably positive, but better repeat or do a Colony-PCR.

16.06.2013

Digest

Investigator: Lukas

Aim: Construction of antibody-vector → Repeat of control digestion.

1 µl DNA template
0,3 µl EcoRI-HF
0,3 µl PstI-HF
1 µl Cut Smart buffer
7,4 µl bidest. H2O

Templates: PSB1A3 iBB486476315 and pSB1A3 iBB48647631 K3 (comparison of size).

The samples were incubated for 1 h at 37 °C.

Gel electrophoresis

Gel substances

1% Agarose gel
10 µl RedSafe per 50 ml gel
6 µl GeneRuler™ 1kb plus DNA Ladder (Thermo Scientific)
6x loading buffer (for samples)

Lane	Content	Expectations
2+3	iBB48647631	4157 bp
4-7	iBB486476315	TBA bp
		Vector pSB1A3: 2150 bp

Too much DNA used, will be repeated as PCR.

17.06.2013

Digest

Investigator: Alex

Aim: Digest of pSB1A3_3+9 and pSB1A3_4+9 with SpeI and PstI, and corresponding inserts pSB1C3_5+4+1+6 and pSB1C3_6+3+1+5 with EcoRI and PstI.

1 µl DNA
0.3 µl Enzyme 1 (SpeI or EcoRI, resp.)
0.3 µl Enzyme 2 (PstI)
1 µl Cut Smart buffer
7.4 µl ddbidest. H2O

The samples were incubated for 1.5 h at 37 °C.

Approaches were unsewed by means of gel electrophoresis, corresponding bands were picked under UV, DNA extraction from gel was performed using DNA Gel Extraction Kit (QIAGEN).

Ligation, Transformation, Plasmid prep

Investigator: Alex

Aim: Assemble the combined bricks 5416 and 6315 into their appropriate vectors pSB1A3_39 and pSB1A3_49, resp.

Insert und plasmid were transformed using a 1:2 ratio. Samples were incubated ON at RT.
Ligation product were transformed into chemical competent DH5α cells (30 min on ice, 90 sec 42 °C, 45 min 37 °C + 900 µl LB).
Transformed cells were spread out on LB Amp culture plates.
Incubation ON at RT.
4 clones were picked for overnight at cultures.
A plasmid preparation was carried out the next day.

22.06.2013

Test digest

Investigator: Alex

Aim: Digest of pSB1A3_395416 and pSB1A3_496315 with EcoRI and PstI.

1 µl Plasmid DNA
0.3 µl EcoRI
0.3 µL PstI
1 µl Cut Smart
7.4 µl ddbidest. H2O

The samples were incubated for 2 h at 37 °C.

Gel electrophoresis

Gel substances

1% Agarose gel
10 µl Ethidium bromide 50 ml gel
4 µl 2-log DNA ladder (New England Biolabs)

Lane	Content	Expectations
upper:
2	iBB6315	1200 + 2000 bp
3	iBB49	1200 + 2000 bp
4-7	iBB496315 K4-1	2400 + 2000 bp
lower:
2	iBB5416	1500 + 2000 bp
3	iBB39	1000 + 2000 bp
4-7	iBB395416 K1-4	2400 + 2000 bp

We didn't receive all expected fragments. All expected fragments in lanes 4-7 were missing. Ligation failed ☹

24.06.2013

PCR

Investigator: Alex

Aim: Check right assembly of BioBrick BBa_K1071005 in the plasmid for antibody production

Volume	Reagent	Temp (°C)	Time
1 µl	pSB1A3_486476315	95	3 min
0.5 µl	Primer fwd	95	30 sec
0.5 µl	Primer rev	60	30 sec	x30
0.5 µl	dNTPs	72	42 sec
0.5 µl	Phusion polymerase	72	5 min
5 µl	Phusion buffer 5x	4	Hold
add 25 µL	ddbidest. H2O

Gel electrophoresis

Gel substances

1% Agarose gel
10 µl Ethidium bromide 50 ml gel
4 µl 2-log DNA ladder (New England Biolabs)

Lane

Content

Expectations

2-5

pSB1A3 iBB486476315 K1-4

~ 6,000 bp for pSB1A3_486476315

280 bp for BBa_K1071005

Each sample contained 2 bands, that means we got all expected bands and it could be assumed that the insertion of BBa_K1071005 was successful.

25.06.2013

Digest

Investigator: Dominik

Aim: Test digest of pSB1A3-iBB39, pSB1A3-iBB49 and pSB1C3-iBB6315 with EcoRI and PstI

1 µl Plasmid (pSB1C3-iBB39,pSB1A3-iBB49 and pSB1C3-iBB6315)
0.3 µl EcoRI
0.3 µl PstI
1 µl CutSmart
7.4 µl ddbidest. H2O

The samples were incubated for 1 h at 37 °C.

Worked as expected.

26.06.2013

Digest

Investigator: Dominik

Aim: Construction of the plasmids pSB1A3-iBB10+9, pSB1A3-iBB11+9, pSB1A3-iBB12+9 and pSB1A3-iBB13+9

1 µl Plasmid (pSB1C3-iBB10, pSB1C3-iBB11, pSB1C3-iBB12 and pSB1C3-iBB13)
0.5 µl EcoRI
0.5 µl SpeI
2 µl CutSmart
12 µl ddbidest. H2O

1 µl Plasmid (pSB1C3-iBB9)
0.5 µl XbaI
0.5 µl Pst/I
2 µl CutSmart
12 µl ddbidest. H2O

1 µl Plasmid (pSB1A3)
0.5 µl EcoRI
0.5 µl PstI
2 µl CutSmart
12 µl ddbidest. H2O

The samples were incubated for 1.5 h at 37 °C.

All expected fragments were present. The relevant fragments were cut from the gel and then purified via QIAgen Gel Extraction Kit (Qiagen).

Ligation

Investigator: Dominik

Aim: Assemble the biobricks iBB10+iBB9, iBB11+iBB9, iBB12+iBB9 and iBB13+iBB9 into the vector pSB1A3.

2 µl vector DNA (pSB1A3)
2 µl insert 1 DNA (iBB9)
2 µl insert 2 DNA (iBB10, iBB11, iBB12 or iBB13)
2 µl 10x T4 DNA ligase buffer
2 µl T4 DNA ligase
2 µl ddbidest. H2O

The samples were incubated overnight at room temperature.

27.06.2013

Transformation

Investigator: Dominik

Aim: Transformation of E. coli DH5α with the plasmid DNA pSB1A3-iBB10+iBB9, pSB1A3-iBB11+iBB9, pSB1A3-iBB12+iBB9 and pSB1A3-iBB13+iBB9.

The chemo competent E. coli DH5α cells were transformed with the plasmids pSB1A3-iBB10+iBB9, pSB1A3-iBB11+iBB9,pSB1A3-iBB12+iBB9 and pSB1A3-iBB13+iBB9 DNA and plated on LB-Amp-plates.
The plates were incubated over night at 37 °C.

Inoculation

Investigator: Dominik

Aim: inoculation of colonies for plasmid preparation

4 colonies of pSB1A3-iBB395146 and 4 colonies of pSB1A3-iBB496315 were inoculated in 5 ml LB medium containing ampicillin.

Digest

Investigator: Alex

Aim: Digest of pSB1A3-iBB395416 and pSB1A3-iBB496315 with EcoRI and PstI.

1 µl Plasmid DNA
0.3 µl EcoRI
0.3 µl PstI
1 µl Cut Smart
7.4 µl ddbidest. H2O

The samples were incubated for 2 h at 37 °C.

Gel electrophoresis

Gel substances

1% Agarose gel
10 µl Ethidium bromide in 50 ml gel
4 µl 2-log DNA ladder (New England Biolabs)

Lane	Content	Expectations
upper:
2-5	iBB395416 K1-4	2400 + 2000 bp
6	iBB39	1000 + 2000 bp
7	iBB6416	1400 + 2000 bp
lower:
2-6	iBB496315 K1-5	2400 + 2000 bp
7	iBB49	1200 + 2000 bp

All clones show a double band above 2000 bp.

We received all expected fragments. The success of the ligation should be double-checked by means of PCR.

@@ Line 1: / Line 1: @@
 {{:Team:Marburg/Template:Header}}
 {{:Team:Marburg/Template:ContentTitleNav}}
-Notebook: June
+Notebook: June <html><a href="https://2013.igem.org/Team:Marburg/Notebook:July"><img src="https://static.igem.org/mediawiki/2013/7/71/Mr-igem-next-arrow.png" style="float:right;margin-left:5px !important;" alt="Next"></a>&nbsp;<a href="https://2013.igem.org/Team:Marburg/Notebook:Mai"><img src="https://static.igem.org/mediawiki/2013/1/13/Mr-igem-previous-arrow.png" alt="Previous" style="float:right;"></a></html>
-{{:Team:Marburg/Template:ContentStartNav}}
+{{:Team:Marburg/Template:ContentStartNav}}<html>
-<html>
-<div class="notebooky-entry">
-<h2 class="title">
-	<a name="Anmerkung">Anmerkung</a>
-</h2>
-<p>Die Teile von Dominik müssen unbedingt noch überarbeitet werden!</p>
-</div>
 <div class="notebooky-entry">
 <h2 class="title">
@@ Line 30: / Line 17: @@
      <div class="aim">
 		<span class="aim">Aim:</span>
-		<span class="aim-desc">Construction of P<sub>NR</sub>-test-vector &rarr; digestion of combined 2 BioBricks.</span>
+		<span class="aim-desc">Construction of P<sub>NR</sub>-test-vector &rarr; Digestion of combined 2 BioBricks.</span>
 	</div>
 	<div class="exp-content">
@@ Line 38: / Line 25: @@
 			<li>0,5 µl enzyme 2</li>
 			<li>2 µl Cut Smart buffer</li>
-			<li>7 µl H2O</li>
+			<li>7 µl bidest. H2O</li>
 		</ul>
 		<p>Samples:</p>
 		<p>Digestion of pSB1A3 iBB1+5 with <i>Xba</i>I and <i>Pst</i>I-HF</p>
 		<p>Digestion of pSB1A3 iBB6+3 with <i>EcoR</i>I-HF and <i>Spe</i>I-HF.</p>
-		<p>The samples were incubated for 1 h at 37° C.</p>
+		<p>The samples were incubated for 1 h at 37 °C.</p>
-		<table class="gel digest">
+		<p>They were purified via QIAgen Gel Extraction Kit (Qiagen). Elution into 20 µl bidest. H2O.</p>
-			<colgroup>
-				<col width="50%" />
-				<col width="50%" />
-			</colgroup>
-		<thead>
-			<tr>
-				<th colspan="2" class="title">Gel electrophoresis</th>
-			</tr>
-		</thead>
-		<tbody>
-			<tr>
-				<td>
-					<img src="beispiel.png" width="50%" alt="gel-electrophoresis-image" />
-				</td>
-				<td>
-					<p>
-					<span class="gel-elc">Gel substances</span>
-					<ul class="gel-sub">
-						<li>1% Agarose gel</li>
-						<li>10 µl RedSafe per 50 ml gel</li>
-						<li>6 µl 2-Log DNA Ladder(0.1-10.0 kb, New England Bioloabs)</li>
-						<li>6x loading buffer (for samples)</li>
-						</ul>
-					</p>
-					<p>The relevant fragments were cut from the gel and then purified via “Qiagen Gel Extraction Kit” (Qiagen, Düsseldorf). Elution into 20 µl H2O.</p>
-				</td>
-			</tr>
-		</tbody>
-		</table>
 	</div>
 </fieldset>
@@ Line 95: / Line 53: @@
 			<li>2 µl 10x T4 DNA ligase buffer</li>
 			<li>1 µl T4 DNA ligase</li>
-			<li>ad 20 µl H2O</li>
+			<li>ad 20 µl bidest. H2O</li>
 		</ul>
 		<p>The samples were incubated for 1 h at RT.</p>
@@ Line 113: / Line 71: @@
 	</div>
 	<div class="exp-content">
-		<p>Complete ligation samples were transformed into <i>E. coli</i> DH5&alpha; (30 min ice, 60 sec 42°C, 60 min 37°C + 900 µl LB), plated on LB<sub>CM</sub>, oN 37°C.</p>
+		<p>Complete ligation samples were transformed into <i>E. coli</i> DH5&alpha; (30 min on ice, 60 sec 42 °C, 60 min 37 °C + 900 µl LB), plated on LB<sub>CM</sub>, overnight at 37 °C.</p>
 	</div>
 </fieldset>
@@ Line 134: / Line 92: @@
 			<li>1 µl 10x T4 DNA ligase buffer</li>
 			<li>1 µl T4 DNA ligase</li>
-			<li>ad 10 µl H2O</li>
+			<li>ad 10 µl bidest. H2O</li>
 		</ul>
 		<p>Samples (all <i>EcoR</i>I/<i>Pst</i>I-cut):</p>
-			<p>120 ng eGFP</p>
+			<p>120 ng eGFP (iBB9)</p>
-				<p>60 ng SPTP1</p>
+				<p>60 ng SPTP1 (iBB10)</p>
-				<p>50 ng SPTP2</p>
+				<p>50 ng SPTP2 (iBB11)</p>
-				<p>55 ng SP1</p>
+				<p>55 ng SP1 (iBB12)</p>
-				<p>50 ng SP2</p>
+				<p>50 ng SP2 (iBB13)</p>
 		<p>The samples were incubated for 3 h at RT.</p>
 	</div>
@@ Line 158: / Line 116: @@
 	</div>
 	<div class="exp-content">
-		<p>Complete ligation samples were transformed into <i>E. coli</i> DH5&alpha; (30 min ice, 60 sec 42°C, 60 min 37°C + 900 µl LB), plated on LB<sub>CM</sub>, oN 37°C.</p>
+		<p>Complete ligation samples were transformed into <i>E. coli</i> DH5&alpha; (30 min on ice, 60 sec 42 °C, 60 min 37 °C + 900 µl LB), plated on LB<sub>CM</sub>, overnight at 37 °C.</p>
 	</div>
 </fieldset>
@@ Line 174: / Line 132: @@
 	</div>
 	<div class="exp-content">
-		<p>3 colonies of pSB1A3 iBB1+6 inoculated in 5 ml LB<sub>amp</sub>, 4 colonies of pSB1C3 iBB4864 inoculated in 5 ml LB<sub>CM</sub>, oN 37°C.</p>
+		<p>3 colonies of pSB1A3 iBB1+6 inoculated in 5 ml LB<sub>amp</sub>, 4 colonies of pSB1C3 iBB4864 inoculated in 5 ml LB<sub>CM</sub>, overnight at 37 °C.</p>
 	</div>
 </fieldset>
@@ Line 198: / Line 156: @@
 	</div>
 	<div class="exp-content">
-		<p>Miniprep of the liquid cultures (“QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf)), eluted in 30 µl H2O. 2 Cultures of pSB1C3 iBB4864 discarded (red colour).</p>
+		<p>Miniprep of the liquid cultures (“QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf)), eluted in 30 µl bidest. H2O. 2 Cultures of pSB1C3 iBB4864 discarded (red colour).</p>
 	</div>
 </fieldset>
@@ Line 219: / Line 177: @@
 			<li>0,5 µl <i>Pst</i>I-HF</li>
 			<li>1 µl Cut Smart buffer</li>
-			<li>7 µl H2O</li>
+			<li>7 µl bidest. H2O</li>
 		</ul>
-		<p>The samples were incubated for 2 h at 37° C.</p>
+		<p>The samples were incubated for 2 h at 37 °C.</p>
 		<table class="gel digest">
 			<colgroup>
@@ Line 235: / Line 193: @@
 			<tr>
 				<td>
-					<img src="beispiel.png" width="50%" alt="gel-electrophoresis-image" />
+					<img src="https://static.igem.org/mediawiki/2013/5/5f/Gel_2013-06-04.png" width="100%" alt="gel-electrophoresis-image" />
 				</td>
 				<td>
@@ Line 243: / Line 201: @@
 						<li>1% Agarose gel</li>
 						<li>10 µl RedSafe per 50 ml gel</li>
-						<li>6 µl 2-Log DNA Ladder(0.1-10.0 kb, New England Bioloabs)</li>
+						<li>6 µl 2-Log DNA Ladder (NEB)</li>
 						<li>6x loading buffer (for samples)</li>
 					</ul>
 					</p>
 					<p>
-						<span class="exp">Expectations</span>
+							<table class="gel">
-						<ul class="exp">
+								<colgroup>
-							<li>Vector: 2 kb</li>
+									<col width="10%" />
-							<li>iBB4864: 2580 bp</li>
+									<col width="2%" />
-							<li>iBB1+6: 650 bp</li>
+									<col width="50%" />
-						</ul>
+									<col width="5%" />
+									<col width="33%" />
+								</colgroup>
+								<thead>
+									<th>Lane</th>
+									<th>&nbsp;</th>
+									<th>Content</th>
+									<th>&nbsp;</th>
+									<th>Expectations</th>
+								</thead>
+								<tr>
+									<td>2+3</td>
+									<th>&nbsp;</th>
+									<td>iBB4864 K1-2</td>
+									<th>&nbsp;</th>
+									<td>2580 bp</td>
+								</tr>
+								<tr>
+									<td>4</td>
+									<th>&nbsp;</th>
+									<td>empty</td>
+									<th>&nbsp;</th>
+									<td></td>
+								<tr>
+									<td>5-7</td>
+									<th>&nbsp;</th>
+									<td>iBB16 K3-5</td>
+									<th>&nbsp;</th>
+									<td>650 bp</td>
+								</tr>
+							</table>
 					</p>
-					<p>All positive.</p>
 				</td>
 			</tr>
@@ Line 280: / Line 267: @@
 			<li>0,5 µl enzyme 2</li>
 			<li>2 µl Cut Smart buffer</li>
-			<li>7 µl H2O</li>
+			<li>7 µl bidest. H2O</li>
 		</ul>
 		<p>Samples:</p>
 		<p>Digestion of pSB1C3 iBB7631 K4 and pSB1A3 iBB16 K4 with <i>Xba</i>I and <i>Pst</i>I-HF</p>
 		<p>Digestion of pSB1C3 iBB4864 K1 and pSB1A3 iBB54 K2 with <i>EcoR</i>I-HF and <i>Spe</i>I-HF.</p>
-		<p>The samples were incubated for 1 h at 37° C.</p>
+		<p>The samples were incubated for 1 h at 37 °C.</p>
-		<table class="gel digest">
+		<p>They were purified via QIAgen Gel Extraction Kit (Qiagen). Elution into 20 µl bidest. H2O.</p>
-			<colgroup>
-				<col width="50%" />
-				<col width="50%" />
-			</colgroup>
-		<thead>
-			<tr>
-				<th colspan="2" class="title">Gel electrophoresis</th>
-			</tr>
-		</thead>
-		<tbody>
-			<tr>
-				<td>
-					<img src="beispiel.png" width="50%" alt="gel-electrophoresis-image" />
-				</td>
-				<td>
-					<p>
-					<span class="gel-elc">Gel substances</span>
-					<ul class="gel-sub">
-						<li>1% Agarose gel</li>
-						<li>10 µl RedSafe per 50 ml gel</li>
-						<li>6 µl 2-Log DNA Ladder(0.1-10.0 kb, New England Bioloabs)</li>
-						<li>6x loading buffer (for samples)</li>
-					</ul>
-					</p>
-					<p>The relevant fragments were cut from the gel and then purified via “Qiagen Gel Extraction Kit” (Qiagen, Düsseldorf). Elution into 20 µl H2O.</p>
-				</td>
-			</tr>
-		</tbody>
-		</table>
 	</div>
 </fieldset>
@@ Line 338: / Line 296: @@
 			<li>1 µl 10x T4 DNA ligase buffer</li>
 			<li>1 µl T4 DNA ligase</li>
-			<li>ad 10 µl H2O</li>
+			<li>ad 10 µl bidest. H2O</li>
 		</ul>
 	<p>Test-vector:</p>
@@ Line 347: / Line 305: @@
 			<li>1 µl 10x T4 DNA ligase buffer</li>
 			<li>1 µl T4 DNA ligase</li>
-			<li>ad 10 µl H2O</li>
+			<li>ad 10 µl bidest. H2O</li>
 		</ul>
-		<p>Both samples were incubated oN at 16° C.</p>
+		<p>Both samples were incubated overnight at 16 °C.</p>
 	</div>
 </fieldset>
@@ Line 365: / Line 323: @@
 	</div>
 	<div class="exp-content">
-		<p>Transformations  (03.06.13) inoculated in 5 ml LB<sub>CM</sub>, oN 37°C.</p>
+		<p>Transformations (03.06.13) inoculated in 5 ml LB<sub>CM</sub>, overnight at 37 °C.</p>
 	</div>
 </fieldset>
@@ Line 389: / Line 347: @@
 	</div>
 	<div class="exp-content">
-		<p>Miniprep of the liquid cultures (“QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf)), eluted in 30 µl H2O.</p>
+		<p>Miniprep of the liquid cultures (“QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf)), eluted in 30 µl bidest. H2O.</p>
 	</div>
 </fieldset>
@@ Line 410: / Line 368: @@
 			<li>0,3 µl <i>Pst</i>I-HF</li>
 			<li>1 µl Cut Smart buffer</li>
-			<li>7,4 µl H2O</li>
+			<li>7,4 µl bidest. H2O</li>
 		</ul>
-		<p>The samples were incubated for 1,5 h at 37° C.</p>
+		<p>The samples were incubated for 1,5 h at 37 °C.</p>
 		<table class="gel digest">
 			<colgroup>
@@ Line 426: / Line 384: @@
 			<tr>
 				<td>
-					<img src="beispiel.png" width="50%" alt="gel-electrophoresis-image" />
+					<img src="https://static.igem.org/mediawiki/2013/f/fd/Gel_2013-06-05_1.png" width="100%" alt="gel-electrophoresis-image" />
 				</td>
 				<td>
@@ Line 434: / Line 392: @@
 						<li>1% Agarose gel</li>
 						<li>10 µl RedSafe per 50 ml gel</li>
-						<li>6 µl 2-Log DNA Ladder(0.1-10.0 kb, New England Bioloabs)</li>
+						<li>6 µl 2-Log DNA Ladder (NEB)</li>
 						<li>6x loading buffer (for samples)</li>
-					</ul>
+						</ul>
 					</p>
 					<p>
-						<span class="exp">Expectations</span>
+							<table class="gel">
-						<ul class="exp">
+								<colgroup>
-							<li>pSB1C3: 2 kb</li>
+									<col width="10%" />
-							<li>iBB9: 780 bp</li>
+									<col width="2%" />
-							<li>iBB10: 228 bp</li>
+									<col width="50%" />
-							<li>iBB11: 147 bp</li>
+									<col width="5%" />
-							<li>iBB12: 123 bp</li>
+									<col width="33%" />
-							<li>iBB13: 108 bp</li>
+								</colgroup>
-							<li>iBB6315: 1200 bp</li>
+								<thead>
-						</ul>
+									<th>Lane</th>
+									<th>&nbsp;</th>
+									<th>Content</th>
+									<th>&nbsp;</th>
+									<th>Expectations</th>
+								</thead>
+								<tr>
+									<td>2-4</td>
+									<th>&nbsp;</th>
+									<td>iBB9 K1-3</td>
+									<th>&nbsp;</th>
+									<td>780 bp</td>
+								</tr>
+								<tr>
+									<td>5</td>
+									<th>&nbsp;</th>
+									<td>empty</td>
+									<th>&nbsp;</th>
+									<td></td>
+								</tr>
+								<tr>
+									<td>6-8</td>
+									<th>&nbsp;</th>
+									<td>iBB10 K1-3</td>
+									<th>&nbsp;</th>
+									<td>228 bp</td>
+								</tr>
+								<tr>
+									<td></td>
+									<th>&nbsp;</th>
+									<td></td>
+									<th>&nbsp;</th>
+									<td>pSB1C3: 2 kb</td>
+								</tr>
+							</table>
+					</p>
+				</td>
+			</tr>
+			<tr>
+				<td>
+					<img src="https://static.igem.org/mediawiki/2013/f/f2/Gel_2013-06-05_2.png" width="100%" alt="gel-electrophoresis-image" />
+				</td>
+				<td>
+					<p>
+							<table class="gel">
+								<colgroup>
+									<col width="10%" />
+									<col width="2%" />
+									<col width="50%" />
+									<col width="5%" />
+									<col width="33%" />
+								</colgroup>
+								<thead>
+									<th>Lane</th>
+									<th>&nbsp;</th>
+									<th>Content</th>
+									<th>&nbsp;</th>
+									<th>Expectations</th>
+								</thead>
+								<tr>
+									<td>2-4</td>
+									<th>&nbsp;</th>
+									<td>iBB11 K1-3</td>
+									<th>&nbsp;</th>
+									<td>147 bp</td>
+								</tr>
+								<tr>
+									<td>5</td>
+									<th>&nbsp;</th>
+									<td>empty</td>
+									<th>&nbsp;</th>
+									<td></td>
+								</tr>
+								<tr>
+									<td>6-8</td>
+									<th>&nbsp;</th>
+									<td>iBB12 K1-3</td>
+									<th>&nbsp;</th>
+									<td>123 bp</td>
+								</tr>
+								<tr>
+									<td></td>
+									<th>&nbsp;</th>
+									<td></td>
+									<th>&nbsp;</th>
+									<td>pSB1C3: 2 kb</td>
+								</tr>
+							</table>
+					</p>
+				</td>
+			</tr>
+			<tr>
+				<td>
+					<img src="https://static.igem.org/mediawiki/2013/5/5a/Gel_2013-06-05_3.png" width="100%" alt="gel-electrophoresis-image" />
+				</td>
+				<td>
+					<p>
+							<table class="gel">
+								<colgroup>
+									<col width="10%" />
+									<col width="2%" />
+									<col width="50%" />
+									<col width="5%" />
+									<col width="33%" />
+								</colgroup>
+								<thead>
+									<th>Lane</th>
+									<th>&nbsp;</th>
+									<th>Content</th>
+									<th>&nbsp;</th>
+									<th>Expectations</th>
+								</thead>
+								<tr>
+									<td>2-4</td>
+									<th>&nbsp;</th>
+									<td>iBB13 K1-3</td>
+									<th>&nbsp;</th>
+									<td>108 bp</td>
+								</tr>
+								<tr>
+									<td>5</td>
+									<th>&nbsp;</th>
+									<td>empty</td>
+									<th>&nbsp;</th>
+									<td></td>
+								</tr>
+								<tr>
+									<td>6-8</td>
+									<th>&nbsp;</th>
+									<td>iBB6315 K1-3</td>
+									<th>&nbsp;</th>
+									<td>1200 bp</td>
+								</tr>
+								<tr>
+									<td></td>
+									<th>&nbsp;</th>
+									<td></td>
+									<th>&nbsp;</th>
+									<td>pSB1C3: 2 kb</td>
+								</tr>
+							</table>
 					</p>
-					<p>Problems with the TBE-buffer %rarr; Longer time with lower volt.</p>
 				</td>
 			</tr>
@@ Line 470: / Line 567: @@
 	</div>
 	<div class="exp-content">
-		<p>Ligations (04.06.13) were transformed into <i>E. coli</i> DH5&alpha; (30 min ice, 60 sec 42°C, 60 min (1C3)/40 min (1A3) 37°C + 900 µl LB), plated on LB<sub>CM</sub> (1C3)/LB<sub>amp</sub> (1A3), oN 37°C.</p>
+		<p>Ligations (04.06.13) were transformed into <i>E. coli</i> DH5&alpha; (30 min on ice, 60 sec 42 °C, 60 min (1C3)/40 min (1A3) 37 °C + 900 µl LB), plated on LB<sub>CM</sub> (1C3)/LB<sub>amp</sub> (1A3), overnight at 37 °C.</p>
 	</div>
 </fieldset>
@@ Line 523: / Line 620: @@
 	</div>
 	<div class="exp-content">
-		<p>3 colonies of pSB1C3 iBB5416 inoculated in 5 ml LB<sub>CM</sub>, 4 colonies of pSB1A3 iBB48647631 inoculated in 5 ml LB<sub>amp</sub>, oN 37°C.</p>
+		<p>3 colonies of pSB1C3 iBB5416 inoculated in 5 ml LB<sub>CM</sub>, 4 colonies of pSB1A3 iBB48647631 inoculated in 5 ml LB<sub>amp</sub>, overnight at 37 °C.</p>
 	</div>
 </fieldset>
@@ Line 547: / Line 644: @@
 	</div>
 	<div class="exp-content">
-		<p>Miniprep of the liquid cultures (“QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf)), eluted in 30 µl H2O.</p>
+		<p>Miniprep of the liquid cultures (“QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf)), eluted in 30 µl bidest. H2O.</p>
 	</div>
 </fieldset>
@@ Line 568: / Line 665: @@
 			<li>0,3 µl <i>Pst</i>I-HF</li>
 			<li>1 µl Cut Smart buffer</li>
-			<li>7,4 µl H2O</li>
+			<li>7,4 µl bidest. H2O</li>
 		</ul>
-		<p>The samples were incubated for 1 h at 37° C.</p>
+		<p>The samples were incubated for 1 h at 37 °C.</p>
 		<table class="gel digest">
 			<colgroup>
@@ Line 584: / Line 681: @@
 			<tr>
 				<td>
-					<img src="beispiel.png" width="50%" alt="gel-electrophoresis-image" />
+					<img src="https://static.igem.org/mediawiki/2013/8/8b/Gel_2013-06-07.png" width="100%" alt="gel-electrophoresis-image" />
 				</td>
 				<td>
@@ Line 592: / Line 689: @@
 						<li>1% Agarose gel</li>
 						<li>10 µl RedSafe per 50 ml gel</li>
-						<li>6 µl 2-Log DNA Ladder(0.1-10.0 kb, New England Bioloabs)</li>
+						<li>6 µl 2-Log DNA Ladder (NEB)</li>
 						<li>6x loading buffer (for samples)</li>
 					</ul>
 					</p>
 					<p>
-						<span class="exp">Expectations</span>
+							<table class="gel">
-						<ul class="exp">
+								<colgroup>
-							<li>pSB1C3: 2070 bp</li>
+									<col width="10%" />
-							<li>iBB5416: 1500 bp</li>
+									<col width="2%" />
-							<li>pSB1A3: 2150 bp</li>
+									<col width="50%" />
-							<li>iBB48647631: 4157 bp</li>
+									<col width="5%" />
-						</ul>
+									<col width="33%" />
+								</colgroup>
+								<thead>
+									<th>Lane</th>
+									<th>&nbsp;</th>
+									<th>Content</th>
+									<th>&nbsp;</th>
+									<th>Expectations</th>
+								</thead>
+								<tr>
+									<td>2-4</td>
+									<th>&nbsp;</th>
+									<td>iBB5416 K1-3</td>
+									<th>&nbsp;</th>
+									<td>
+										<p>pSB1C3: 2070 bp</p>
+										<p>iBB5416: 1500 bp</p>
+									</td>
+								</tr>
+								<tr>
+									<td>5-8</td>
+									<th>&nbsp;</th>
+									<td>iBB48647631 K1-4</td>
+									<th>&nbsp;</th>
+									<td>
+										<p>pSB1A3: 2150 bp</p>
+										<p>iBB48647631: 4157 bp</p>
+									</td>
+								</tr>
+							</table>
 					</p>
-					<p>All positive.</p>
 				</td>
 			</tr>
@@ Line 638: / Line 763: @@
 			<li>0,5 µl enzyme 2</li>
 			<li>2,2 µl Cut Smart buffer</li>
-			<li>20 µl H2O</li>
+			<li>20 µl bidest. H2O</li>
 		</ul>
 		<p>Samples:</p>
@@ Line 644: / Line 769: @@
 		<p>Digestion of 5 µl pSB1C3 iBB5 and iBB9 with <i>Xba</i>I and <i>Pst</i>I-HF</p>
 		<p>Digestion of 4 µl pSB1C3 iBB3 and iBB4 with <i>EcoR</i>I-HF and <i>Spe</i>I-HF.</p>
-		<p>The samples were incubated for 1 h at 37° C.</p>
+		<p>The samples were incubated for 1 h at 37 °C.</p>
-		<table class="gel digest">
+		<p>They were purified via QIAgen Gel Extraction Kit (Qiagen). Elution into 20 µl bidest. H2O.</p>
-			<colgroup>
-				<col width="50%" />
-				<col width="50%" />
-			</colgroup>
-		<thead>
-			<tr>
-				<th colspan="2" class="title">Gel electrophoresis</th>
-			</tr>
-		</thead>
-		<tbody>
-			<tr>
-				<td>
-					<img src="beispiel.png" width="50%" alt="gel-electrophoresis-image" />
-				</td>
-				<td>
-					<p>
-					<span class="gel-elc">Gel substances</span>
-					<ul class="gel-sub">
-						<li>1% Agarose gel</li>
-						<li>10 µl RedSafe per 50 ml gel</li>
-						<li>6 µl 2-Log DNA Ladder(0.1-10.0 kb, New England Bioloabs)</li>
-						<li>6x loading buffer (for samples)</li>
-					</ul>
-					</p>
-					<p>The relevant fragments were cut from the gel and then purified via “Qiagen Gel Extraction Kit” (Qiagen, Düsseldorf). Elution into 20 µl H2O.</p>
-				</td>
-			</tr>
-		</tbody>
-		</table>
 	</div>
 </fieldset>
@@ Line 690: / Line 786: @@
 	</div>
 	<div class="exp-content">
-		<p>Ligation of pSB1A3 iBB48647631 with iBB5, pSB1A3 iBB48647631 with iBB3 and iBB9, pSB1A3 iBB48647631 with iBB4 and iBB9; oN 16°C</p>
+		<p>Ligation of pSB1A3 iBB48647631 with iBB5, pSB1A3 iBB48647631 with iBB3 and iBB9, pSB1A3 iBB48647631 with iBB4 and iBB9; overnight at 16°C</p>
 	</div>
 </fieldset>
@@ Line 714: / Line 810: @@
 	</div>
 	<div class="exp-content">
-		<p>Transformation of the ligations into <i>E. coli</i> DH5&alpha; (30 min ice, 60 sec 42°C, 25 min 37°C + 900 µl LB), plated on LB<sub>amp</sub>, oN 37°C.</p>
+		<p>Transformation of the ligations into <i>E. coli</i> DH5&alpha; (30 min on ice, 60 sec 42 °C, 25 min 37 °C + 900 µl LB), plated on LB<sub>amp</sub>, overnight at 37 °C.</p>
 	</div>
 </fieldset>
@@ Line 738: / Line 834: @@
 	</div>
 	<div class="exp-content">
-		<p>4 colonies of each transformation inoculated in 4 ml LB<sub>amp</sub>, oN 37°C.</p>
+		<p>4 colonies of each transformation inoculated in 4 ml LB<sub>amp</sub>, overnight at 37 °C.</p>
 	</div>
 </fieldset>
@@ Line 762: / Line 858: @@
 	</div>
 	<div class="exp-content">
-		<p>Miniprep of the liquid cultures (“QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf)), eluted in 30 µl H2O.</p>
+		<p>Miniprep of the liquid cultures (“QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf)), eluted in 30 µl bidest. H2O.</p>
 	</div>
 </fieldset>
@@ Line 783: / Line 879: @@
 			<li>0,3 µl <i>Pst</i>I-HF</li>
 			<li>1 µl Cut Smart buffer</li>
-			<li>7,4 µl H2O</li>
+			<li>7,4 µl bidest. H2O</li>
 		</ul>
-		<p>The samples were incubated for 1 h at 37° C.</p>
+		<p>The samples were incubated for 1 h at 37 °C.</p>
 		<table class="gel digest">
 			<colgroup>
@@ Line 799: / Line 895: @@
 			<tr>
 				<td>
-					<img src="beispiel.png" width="50%" alt="gel-electrophoresis-image" />
+					<img src="https://static.igem.org/mediawiki/2013/2/22/Gel_2013-06-13_1.png" width="100%" alt="gel-electrophoresis-image" />
 				</td>
 				<td>
@@ Line 807: / Line 903: @@
 						<li>1% Agarose gel</li>
 						<li>10 µl RedSafe per 50 ml gel</li>
-						<li>6 µl 2-Log DNA Ladder(0.1-10.0 kb, New England Bioloabs)</li>
+						<li>6 µl 2-Log DNA Ladder (NEB)</li>
 						<li>6x loading buffer (for samples)</li>
 					</ul>
 					</p>
 					<p>
-						<span class="exp">Expectations</span>
+							<table class="gel">
-						<ul class="exp">
+								<colgroup>
-							<li>pSB1A3: 2150 bp</li>
+									<col width="10%" />
-							<li>iBB48647631: 4157 bp</li>
+									<col width="2%" />
-							<li>iBB486476315: 4404 bp</li>
+									<col width="50%" />
-							<li>iBB49: 1260 bp</li>
+									<col width="5%" />
-							<li>iBB39: 1030 bp</li>
+									<col width="33%" />
-						</ul>
+								</colgroup>
+								<thead>
+									<th>Lane</th>
+									<th>&nbsp;</th>
+									<th>Content</th>
+									<th>&nbsp;</th>
+									<th>Expectations</th>
+								</thead>
+								<tr>
+									<td>upper:</td>
+								</tr>
+								<tr>
+									<td>2+3</td>
+									<th>&nbsp;</th>
+									<td>iBB49 K3-4</td>
+									<th>&nbsp;</th>
+									<td>1260 bp</td>
+								</tr>
+								<tr>
+									<td>4-7</td>
+									<th>&nbsp;</th>
+									<td>iBB39 K1-4</td>
+									<th>&nbsp;</th>
+									<td>1030 bp</td>
+								</tr>
+								<tr>
+									<td>lower:</td>
+								</tr>
+								<tr>
+									<td>2</td>
+									<th>&nbsp;</th>
+									<td>iBB49647631</td>
+									<th>&nbsp;</th>
+									<td>4157 bp</td>
+								</tr>
+								<tr>
+									<td>3-6</td>
+									<th>&nbsp;</th>
+									<td>iBB496476315 K1-4</td>
+									<th>&nbsp;</th>
+									<td>4404 bp</td>
+								</tr>
+								<tr>
+									<td>7+8</td>
+									<th>&nbsp;</th>
+									<td>iBB49 K1-2</td>
+									<th>&nbsp;</th>
+									<td>1260 bp</td>
+								</tr>
+								<tr>
+									<td></td>
+									<th>&nbsp;</th>
+									<td></td>
+									<th>&nbsp;</th>
+									<td>Vector pSB1A3: 2150 bp</td>
+								</tr>
+							</table>
 					</p>
-					<p>For better separation another 30 min.</p>
+					<p>IBB48647631 and iBB486476315 probably positive, but better repeat or do a Colony-PCR.</p>
-				</td>
-			</tr>
-			<tr>
-				<td>
-					<img src="beispiel.png" width="50%" alt="gel-electrophoresis-image" />
-				</td>
-				<td>
-					<p>Probably positive, but better repeat or do a Colony-PCR.</p>
 				</td>
 			</tr>
@@ Line 862: / Line 1,006: @@
 			<li>0,3 µl <i>Pst</i>I-HF</li>
 			<li>1 µl Cut Smart buffer</li>
-			<li>7,4 µl H2O</li>
+			<li>7,4 µl bidest. H2O</li>
 		</ul>
 		<p>Templates: PSB1A3 iBB486476315 and pSB1A3 iBB48647631 K3 (comparison of size).</p>
-		<p>The samples were incubated for 1 h at 37° C.</p>
+		<p>The samples were incubated for 1 h at 37 °C.</p>
 		<table class="gel digest">
 			<colgroup>
@@ Line 879: / Line 1,023: @@
 			<tr>
 				<td>
-					<img src="beispiel.png" width="50%" alt="gel-electrophoresis-image" />
+					<img src="https://static.igem.org/mediawiki/2013/3/38/Gel_2013-06-16.png" width="100%" alt="gel-electrophoresis-image" />
 				</td>
 				<td>
@@ Line 892: / Line 1,036: @@
 					</p>
 					<p>
-						<span class="exp">Expectations</span>
+							<table class="gel">
-						<ul class="exp">
+								<colgroup>
-							<li>pSB1A3: 2150 bp</li>
+									<col width="10%" />
-							<li>iBB48647631: 4157 bp</li>
+									<col width="2%" />
-							<li>iBB486476315: 4404 bp</li>
+									<col width="50%" />
-						</ul>
+									<col width="5%" />
+									<col width="33%" />
+								</colgroup>
+								<thead>
+									<th>Lane</th>
+									<th>&nbsp;</th>
+									<th>Content</th>
+									<th>&nbsp;</th>
+									<th>Expectations</th>
+								</thead>
+								<tr>
+									<td>2+3</td>
+									<th>&nbsp;</th>
+									<td>iBB48647631</td>
+									<th>&nbsp;</th>
+									<td>4157 bp</td>
+								</tr>
+								<tr>
+									<td>4-7</td>
+									<th>&nbsp;</th>
+									<td>iBB486476315</td>
+									<th>&nbsp;</th>
+									<td>TBA bp</td>
+								</tr>
+								<tr>
+									<td></td>
+									<th>&nbsp;</th>
+									<td></td>
+									<th>&nbsp;</th>
+									<td>Vector pSB1A3: 2150 bp</td>
+								</tr>
+							</table>
 					</p>
 					<p>Too much DNA used, will be repeated as PCR.</p>
@@ Line 928: / Line 1,103: @@
 		<ul class="digest">
   			<li>1 µl DNA</li>
-			<li>0.3 µl Enzyme 1 (SpeI or EcoRI, resp.)</li>
+			<li>0.3 µl Enzyme 1 (<i>Spe</i>I or <i>EcoR</i>I, resp.)</li>
-			<li>0.3 µl Enzyme 2 (PstI)</li>
+			<li>0.3 µl Enzyme 2 (<i>Pst</i>I)</li>
-			<li>1 µl Cut Smart</li>
+			<li>1 µl Cut Smart buffer</li>
-			<li>7.4 µl ddH2O</li>
+			<li>7.4 µl ddbidest. H2O</li>
 		</ul>
-		<p>The samples were incubated for 1.5 h at 37° C.</p>
+		<p>The samples were incubated for 1.5 h at 37 °C.</p>
-                <p>Approaches were unsewed by means of gel electrophoresis, corresponding bands were picked under UV, DNA extraction from gel was performed using DNA Gel Extraction Kit (<i>QIAGEN</i>).</p>
+		<p>Approaches were unsewed by means of gel electrophoresis, corresponding bands were picked under UV, DNA extraction from gel was performed using DNA Gel Extraction Kit (<i>QIAGEN</i>).</p>
 </fieldset>
@@ Line 946: / Line 1,120: @@
      <div class="aim">
 		<span class="aim">Aim:</span>
-		<span class="aim-desc">Assemble the combined bricks 5416 and 6315 in their appropriate vectors pSB1A3_39 and pSB1A3_49, resp.</span>
+		<span class="aim-desc">Assemble the combined bricks 5416 and 6315 into their appropriate vectors pSB1A3_39 and pSB1A3_49, resp.</span>
 	</div>
 	<div class="exp-content">
 		<ul class="lig">
 			<li>Insert und plasmid were transformed using a 1:2 ratio. Samples were incubated ON at RT.</li>
-			<li>Ligation product were transformed into chemical competent DH5α cells (30 min ice, 90 sec 42°C, 45 min 37°C + 900 µl LB).</li>
+			<li>Ligation product were transformed into chemical competent DH5α cells (30 min on ice, 90 sec 42 °C, 45 min 37 °C + 900 µl LB).</li>
 			<li>Transformed cells were spread out on LB Amp culture plates.</li>
 			<li>Incubation ON at RT.</li>
-                         <li>4 clones were picked for overnight cultures.</li>
+                         <li>4 clones were picked for overnight at cultures.</li>
                          <li>A plasmid preparation was carried out the next day.</li>
 		</ul>
@@ Line 982: / Line 1,156: @@
 			<li>0.3 µL PstI</li>
 			<li>1 µl Cut Smart</li>
-			<li>7.4 µl ddH2O</li>
+			<li>7.4 µl ddbidest. H2O</li>
 		</ul>
-		<p>The samples were incubated for 2 h at 37° C.</p>
+		<p>The samples were incubated for 2 h at 37 °C.</p>
 		<table class="gel digest">
 			<colgroup>
@@ Line 998: / Line 1,172: @@
 			<tr>
 				<td>
-					<img src="beispiel.png" width="50%" alt="gel-electrophoresis-image" />
+					<img src="https://static.igem.org/mediawiki/2013/5/53/Gel_2013-06-17_2.png" width="100%" alt="gel-electrophoresis-image" />
 				</td>
 				<td>
@@ Line 1,006: / Line 1,180: @@
 						<li>1% Agarose gel</li>
 						<li>10 µl Ethidium bromide 50 ml gel</li>
-						<li>4 µl 2-log DNA ladder</li>
+						<li>4 µl 2-log DNA ladder (New England Biolabs)</li>
 					</ul>
 					</p>
 					<p>
-						<span class="exp">Expectations (upper gel)</span>
+							<table class="gel">
-						<ul class="exp">
+								<colgroup>
-							<li>Lane 2: 1,200 + 2,000 bp</li>
+									<col width="10%" />
-							<li>Lane 3: 1,200 + 2,000 bp</li>
+									<col width="2%" />
-							<li>Lane 4-7: 2,400 + 2,000 bp</li>
+									<col width="50%" />
-						</ul></p>
+									<col width="5%" />
-                                                <p><span class="exp">Expectations (lower gel)</span>
+									<col width="33%" />
-						<ul class="exp">
+								</colgroup>
-							<li>Lane 2: 1,500 + 2,000 bp</li>
+								<thead>
-							<li>Lane 3: 1,000 + 2,000 bp</li>
+									<th>Lane</th>
-							<li>Lane 4-7: 2,400 + 2,000 bp</li>
+									<th>&nbsp;</th>
-						</ul></p>
+									<th>Content</th>
+									<th>&nbsp;</th>
+									<th>Expectations</th>
+								</thead>
+								<tr>
+									<td>upper:</td>
+								</tr>
+								<tr>
+									<td>2</td>
+									<th>&nbsp;</th>
+									<td>iBB6315</td>
+									<th>&nbsp;</th>
+									<td>1200 + 2000 bp</td>
+								</tr>
+								<tr>
+									<td>3</td>
+									<th>&nbsp;</th>
+									<td>iBB49</td>
+									<th>&nbsp;</th>
+									<td>1200 + 2000 bp</td>
+								</tr>
+								<tr>
+								<tr>
+									<td>4-7</td>
+									<th>&nbsp;</th>
+									<td>iBB496315 K4-1</td>
+									<th>&nbsp;</th>
+									<td>2400 + 2000 bp</td>
+								</tr>
+								<tr>
+									<td>lower:</td>
+								</tr>
+								<tr>
+									<td>2</td>
+									<th>&nbsp;</th>
+									<td>iBB5416</td>
+									<th>&nbsp;</th>
+									<td>1500 + 2000 bp</td>
+								</tr>
+								<tr>
+									<td>3</td>
+									<th>&nbsp;</th>
+									<td>iBB39</td>
+									<th>&nbsp;</th>
+									<td>1000 + 2000 bp</td>
+								</tr>
+								<tr>
+									<td>4-7</td>
+									<th>&nbsp;</th>
+									<td>iBB395416 K1-4</td>
+									<th>&nbsp;</th>
+									<td>2400 + 2000 bp</td>
+								</tr>
+							</table>
 					</p>
 				</td>
@@ Line 1,027: / Line 1,254: @@
 			<tr>
 				<td colspan="2" class="gel-fazit">
-					<p>We didn’t receive all expected fragments. All expected fragments in lanes 4-7 were missing. Ligation failed :(</p>
+					<p>We didn't receive all expected fragments. All expected fragments in lanes 4-7 were missing. Ligation failed &#x2639;</p>
 				</td>
 			</tr>
@@ Line 1,050: / Line 1,277: @@
      <div class="aim">
 		<span class="aim">Aim:</span>
-		<span class="aim-desc">Check right assembly of biobrick BBa_K1071005 in the plasmid for antibody production</span>
+		<span class="aim-desc">Check right assembly of BioBrick BBa_K1071005 in the plasmid for antibody production</span>
 	</div>
 	<div class="exp-content">
@@ Line 1,114: / Line 1,341: @@
                          <tr>
 				<td>add 25 µL</td>
-				<td>ddH2O</td>
+				<td>ddbidest. H2O</td>
 				<td>&nbsp;</td>
 			</tr>
@@ Line 1,133: / Line 1,360: @@
 			<tr>
 				<td>
-					<img src="beispiel.png" width="50%" alt="gel-electrophoresis-image" />
+					<img src="https://static.igem.org/mediawiki/2013/5/58/Gel_2013-06-24.png" width="100%" alt="gel-electrophoresis-image" />
 				</td>
 				<td>
@@ Line 1,141: / Line 1,368: @@
 						<li>1% Agarose gel</li>
 						<li>10 µl Ethidium bromide 50 ml gel</li>
-						<li>4 µl 2-log DNA ladder</li>
+						<li>4 µl 2-log DNA ladder (New England Biolabs)</li>
 					</ul>
 					</p>
 					<p>
-						<span class="exp">Expectations</span>
+							<table class="gel">
-						<ul class="exp">
+								<colgroup>
-							<li>Lane 1: ~ 6,000 bp for pSB1A3_486476315</li>
+									<col width="10%" />
-							<li>Lane 2:     280 bp for BBa_K1071005</li>
+									<col width="2%" />
-						</ul>
+									<col width="50%" />
+									<col width="5%" />
+									<col width="33%" />
+								</colgroup>
+								<thead>
+									<th>Lane</th>
+									<th>&nbsp;</th>
+									<th>Content</th>
+									<th>&nbsp;</th>
+									<th>Expectations</th>
+								</thead>
+								<tr>
+									<td>2-5</td>
+									<th>&nbsp;</th>
+									<td>pSB1A3 iBB486476315 K1-4</td>
+									<th>&nbsp;</th>
+									<td>
+										<p>~ 6,000 bp for pSB1A3_486476315</p>
+										<p>280 bp for BBa_K1071005</p>
+									</td>
+								</tr>
+							</table>
 					</p>
 					<p>Each sample contained 2 bands, that means we got all expected bands and it could be assumed that the insertion of BBa_K1071005 was successful.</p>
@@ Line 1,174: / Line 1,422: @@
      <div class="aim">
 		<span class="aim">Aim:</span>
-		<span class="aim-desc">Test digest of pSB1A3-iBB39, pSB1A3-iBB49 and pSB1C3-iBB6315 with <i>Eco</i>RI and <i>Pst</i>I</span>
+		<span class="aim-desc">Test digest of pSB1A3-iBB39, pSB1A3-iBB49 and pSB1C3-iBB6315 with <i>EcoR</i>I and <i>Pst</i>I</span>
 	</div>
 	<div class="exp-content">
 		<p><ul class="digest">
 			<li>1 µl Plasmid (pSB1C3-iBB39,pSB1A3-iBB49 and pSB1C3-iBB6315)</li>
-			<li>0.3 µl EcoRI</li>
+			<li>0.3 µl <i>EcoR</i>I</li>
-			<li>0.3 µl PstI</li>
+			<li>0.3 µl <i>Pst</i>I</li>
 			<li>1 µl CutSmart</li>
-			<li>7.4 µl ddH2O</li>
+			<li>7.4 µl ddbidest. H2O</li>
 		</ul>
-		<p>The samples were incubated for 1 h at 37° C.</p>
+		<p>The samples were incubated for 1 h at 37 °C.</p>
-		<table class="gel digest">
+		<p>Worked as expected.</p>
-			<colgroup>
-				<col width="50%" />
-				<col width="50%" />
-			</colgroup>
-		<thead>
-			<tr>
-				<th colspan="2" class="title">Gel electrophoresis</th>
-			</tr>
-		</thead>
-		<tbody>
-			<tr>
-				<td>
-					<img src="beispiel.png" width="50%" alt="gel-electrophoresis-image" />
-				</td>
-				<td>
-					<p>
-					<span class="gel-elc">Gel substances</span>
-					<ul class="gel-sub">
-						<li>1% Agarose gel</li>
-						<li>10 µl RedSafe in 50 ml gel</li>
-						<li>x µl Hyper Ladder</li>
-					</ul>
-					</p>
-					<p>
-						<span class="exp">Expectations</span>
-						<ul class="exp">
-							<li>Lane 1: 5300 kbp</li>
-							<li>Lane 2: 3300 kbp</li>
-							<li>Lane 3: 1300 kbp</li>
-						</ul>
-					</p>
-					<p>Worked as expected.</p>
-				</td>
-			</tr>
-		</tbody>
-		</table>
 	</div>
 </fieldset>
@@ Line 1,244: / Line 1,456: @@
 		<p><ul class="digest">
 			<li>1 µl Plasmid (pSB1C3-iBB10, pSB1C3-iBB11, pSB1C3-iBB12 and pSB1C3-iBB13)</li>
-			<li>0.5 µl EcoRI</li>
+			<li>0.5 µl <i>EcoR</i>I</li>
-			<li>0.5 µl SpeI</li>
+			<li>0.5 µl <i>Spe</i>I</li>
 			<li>2 µl CutSmart</li>
-			<li>12 µl ddH2O</li>
+			<li>12 µl ddbidest. H2O</li>
 		</ul>
 		<p><ul class="digest">
 			<li>1 µl Plasmid (pSB1C3-iBB9)</li>
-			<li>0.5 µl XbaI</li>
+			<li>0.5 µl <i>Xba</i>I</li>
-			<li>0.5 µl PstI</li>
+			<li>0.5 µl <i>Pst/</i>I</li>
 			<li>2 µl CutSmart</li>
-			<li>12 µl ddH2O</li>
+			<li>12 µl ddbidest. H2O</li>
 		</ul>
 		<p><ul class="digest">
 			<li>1 µl Plasmid (pSB1A3)</li>
-			<li>0.5 µl EcoRI</li>
+			<li>0.5 µl <i>EcoR</i>I</li>
-			<li>0.5 µl PstI</li>
+			<li>0.5 µl <i>Pst</i>I</li>
 			<li>2 µl CutSmart</li>
-			<li>12 µl ddH2O</li>
+			<li>12 µl ddbidest. H2O</li>
 		</ul>
-		<p>The samples were incubated for 1.5 h at 37° C.</p>
+		<p>The samples were incubated for 1.5 h at 37 °C.</p>
-		<table class="gel digest">
+		<p>All expected fragments were present. The relevant fragments were cut from the gel and then purified via QIAgen Gel Extraction Kit (Qiagen).</p>
-			<colgroup>
-				<col width="50%" />
-				<col width="50%" />
-			</colgroup>
-		<thead>
-			<tr>
-				<th colspan="2" class="title">Gel electrophoresis</th>
-			</tr>
-		</thead>
-		<tbody>
-			<tr>
-				<td>
-					<img src="beispiel.png" width="50%" alt="gel-electrophoresis-image" />
-				</td>
-				<td>
-					<p>
-					<span class="gel-elc">Gel substances</span>
-					<ul class="gel-sub">
-						<li>1% Agarose gel</li>
-						<li>10 µl RedSafe in 50 ml gel</li>
-						<li>x µl Hyper Ladder</li>
-					</ul>
-					</p>
-					<p>
-						<span class="exp">Expectations</span>
-						<ul class="exp">
-							<li>Lane 1: 5300 kbp</li>
-							<li>Lane 2: 3300 kbp</li>
-							<li>Lane 3: 1300 kbp</li>
-						</ul>
-					</p>
-					<p>All expected fragments were present. The relevant fragments were cut from the gel and then purified via “Qiagen Gel Extraction Kit” (Qiagen, Düsseldorf).</p>
-				</td>
-			</tr>
-		</tbody>
-		</table>
 	</div>
 </fieldset>
@@ Line 1,324: / Line 1,500: @@
 			<li>2 µl 10x T4 DNA ligase buffer</li>
 			<li>2 µl T4 DNA ligase</li>
-			<li>2 µl ddH2O</li>
+			<li>2 µl ddbidest. H2O</li>
 		</ul>
 		<p>The samples were incubated overnight at room temperature.</p>
@@ Line 1,348: / Line 1,524: @@
 	<div class="exp-content">
 		<p>The chemo competent <i>E. coli</i> DH5&alpha; cells were transformed with the plasmids pSB1A3-iBB10+iBB9, pSB1A3-iBB11+iBB9,pSB1A3-iBB12+iBB9 and pSB1A3-iBB13+iBB9 DNA and plated on LB-Amp-plates.<br />
-		The plates were incubated over night at 37° C.</p>
+		The plates were incubated over night at 37 °C.</p>
 	</div>
 </fieldset>
@@ Line 1,380: / Line 1,556: @@
 		<ul class="digest">
 			<li>1 µl Plasmid DNA</li>
-			<li>0.3 µl EcoRI</li>
+			<li>0.3 µl <i>EcoR</i>I</li>
-			<li>0.3 µl PstI</li>
+			<li>0.3 µl <i>Pst</i>I</li>
 			<li>1 µl Cut Smart</li>
-			<li>7.4 µl ddH2O</li>
+			<li>7.4 µl ddbidest. H2O</li>
 		</ul>
-		<p>The samples were incubated for 2 h at 37° C.</p>
+		<p>The samples were incubated for 2 h at 37 °C.</p>
 		<table class="gel digest">
 			<colgroup>
@@ Line 1,399: / Line 1,575: @@
 			<tr>
 				<td>
-					<img src="beispiel.png" width="50%" alt="gel-electrophoresis-image" />
+					<img src="https://static.igem.org/mediawiki/2013/5/52/Gel_2013-06-27.png" width="100%" alt="gel-electrophoresis-image" />
 				</td>
 				<td>
@@ Line 1,407: / Line 1,583: @@
 						<li>1% Agarose gel</li>
 						<li>10 µl Ethidium bromide in 50 ml gel</li>
-						<li>4 µl 2-log DNA ladder</li>
+						<li>4 µl 2-log DNA ladder (New England Biolabs)</li>
 					</ul>
 					</p>
 					<p>
-						<span class="exp">Expectations (upper gel)</span>
+							<table class="gel">
-						<ul class="exp">
+								<colgroup>
-							<li>Lane 2-5: 2,400 + 2,000 bp</li>
+									<col width="10%" />
-							<li>Lane 6: 1,000 + 2,000 bp</li>
+									<col width="2%" />
-							<li>Lane 7: 1,400 + 2,000 bp</li>
+									<col width="50%" />
-						</ul></p>
+									<col width="5%" />
-                                                <p><span class="exp">Expectations (lower gel)</span>
+									<col width="33%" />
-						<ul class="exp">
+								</colgroup>
-							<li>Lane 2-7: 2,400 + 2,000 bp</li>
+								<thead>
-							<li>Lane 8: 1,200 + 2,000 bp</li>
+									<th>Lane</th>
-						</ul></p>
+									<th>&nbsp;</th>
+									<th>Content</th>
+									<th>&nbsp;</th>
+									<th>Expectations</th>
+								</thead>
+								<tr>
+									<td>upper:</td>
+								</tr>
+								<tr>
+									<td>2-5</td>
+									<th>&nbsp;</th>
+									<td>iBB395416 K1-4</td>
+									<th>&nbsp;</th>
+									<td>2400 + 2000 bp</td>
+								</tr>
+								<tr>
+									<td>6</td>
+									<th>&nbsp;</th>
+									<td>iBB39</td>
+									<th>&nbsp;</th>
+									<td>1000 + 2000 bp</td>
+								</tr>
+								<tr>
+								<tr>
+									<td>7</td>
+									<th>&nbsp;</th>
+									<td>iBB6416</td>
+									<th>&nbsp;</th>
+									<td>1400 + 2000 bp</td>
+								</tr>
+								<tr>
+									<td>lower:</td>
+								</tr>
+								<tr>
+									<td>2-6</td>
+									<th>&nbsp;</th>
+									<td>iBB496315 K1-5</td>
+									<th>&nbsp;</th>
+									<td>2400 + 2000 bp</td>
+								</tr>
+								<tr>
+									<td>7</td>
+									<th>&nbsp;</th>
+									<td>iBB49</td>
+									<th>&nbsp;</th>
+									<td>1200 + 2000 bp</td>
+								</tr>
+							</table>
 					</p>
-					<p>All clones show a double band above 2,000 bp.</p>
+					<p>All clones show a double band above 2000 bp.</p>
 				</td>
 			</tr>

Team:Marburg/Notebook:June

From 2013.igem.org

Latest revision as of 11:14, 27 October 2013

Notebook: June