From 2013.igem.org

Notebook: April

02.04.2013

Ligation

Investigator: Christian

Aim: Construction of BioBricks → Ligation of iBB2 into pJet1.2.

1 µl pJet1.2 (provided already cut)
25 µl PCR-product CAT-cassette
3 µl 10x T4 DNA ligase buffer
1 µl T4 DNA ligase

The samples are incubated for 2 h at RT.

Transformation

Investigator: Christian

Aim: Construction of BioBricks → Transformation of pJet1.2 iBB2.

Transformation into E. coli DH5α (30 min ice, 60 sec 42°C, 60 sec ice, 45 min 37°C + 1 ml LB), plated on LB_amp, overnight at 37°C.

03.04.2013

PCR

Investigator: Christian

Aim: Construction of BioBricks → Control of pJet1.2 iBB2 via colony-PCR.

Volume	Reagent	Temp (°C)	Time
1 µl	Phusion polymerase	98	3 min
1 µl	Primer fw	98	30 sec
1 µl	Primer rv	65	30 sec	x26
10 µl	Template	72	1 min
1 µl	dNTPs	72	7 min
10 µl	5x buffer	4	Hold
26 µl	bidest. H2O

Gel electrophoresis

Gel substances

1% Agarose gel
10 µl RedSafe in 50 ml gel
6 µl GeneRuler™ 2-log DNA Ladder (NEB)
6x loading buffer (for samples)

Lane		Content		Expectations
2-8		Colony PCR iBB2		717 bp

We receive all expected fragments.

Colony PCR is successful.

Inoculation

Investigator: Christian

Aim: Construction of BioBricks → Inoculation of positive pJet1.2 iBB2.

Colonies 1-3 + 5 inoculated in 5 ml LB_amp, overnight at 37°C.

04.04.2013

Miniprep

Investigator: Christian

Aim: Construction of BioBricks → Miniprep of pJet1.2 iBB2.

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

PCR

Investigator: Christian

Aim: Construction of BioBricks → Deletion of restriction sites in iBB2 via mutagenesis PCR.

Mutagenesis PCR for point mutation in PvuII-site of K3 and K5.

Volume	Reagent	Temp (°C)	Time
1 µl	Phusion polymerase	95	3 min
1,5 µl	Primer 13 (10 pmol/µl)	95	30 sec
1,5 µl	Primer 14 (10 pmol/µl)	58	45 sec	x19
1 µl	Template	72	4 min
1 µl	dNTPs	72	6 min
5 µl	10x buffer	4	Hold
ad 50 µl	bidest. H2O

PCR

Investigator: Christian

Aim: Construction of BioBricks → Deletion of restriction sites in iBB2 via mutagenesis PCR.

Mutagenesis PCR for point mutation in EcoRI-site of K3 and K5.

Volume	Reagent	Temp (°C)	Time
1 µl	Phusion polymerase	95	3 min
1,5 µl	Primer 15 (10 pmol/µl)	95	30 sec
1,5 µl	Primer 16 (10 pmol/µl)	63	45 sec	x19
1 µl	Template	72	4 min
1 µl	dNTPs	72	6 min
5 µl	10x buffer	4	Hold
ad 50 µl	bidest. H2O

05.04.2013

Transformation

Investigator: Christian

Aim: Construction of BioBricks → Transformation of mutagenised pJet1.2 iBB2 into E. coli.

Transformation of products of the second PCR into E. coli DH5α ( 30 min ice, 60 sec 42°C, 60 sec ice, 45 min 37°C + 1 ml LB), plated on LB_amp, overnight at 37°C.

08.04.2013

PCR

Investigator: Christian

Aim: Construction of BioBricks → Repeat of mutagenesis of pJet1.2 iBB2 and adding of pre- and suffix of iBB1, iBB6, iBB7 and iBB8.

PCR of iBB1 (pre- and suffix), iBB6 (pre- and suffix), and iBB2 (mutagenesis of PvuII-site), second PCR of iBB2 (mutagenesis of EcoRI-site), iBB7 (pre- and suffix) and iBB8 (pre- and suffix). All done with analog mix and program. Primers vary.

Volume	Reagent	Temp (°C)	Time
1 µl	Phusion polymerase	95	3 min
1 µl	Primer fw	95	30 sec
1 µl	Primer rv	65	45 sec	x19
1 µl	Template	72	4 min
1 µl	dNTPs	72	6 min
5 µl	10x buffer	4	Hold
10 µl	bidest. H2O

Digest

Investigator: Christian

Aim: Preparation of pSB1C3 → Digestion of pSB1C3 J04450.

Digestion of pSB1C3 J04450 (2012) with EcoRI and PstI.

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		pPha-NR cut with EcoRI and PstI.		2868 bp + 992 bp
3		pPha-T1 cut with EcoRI and PstI.		3554 bp + 541 bp

Lanes 2 and 3 contained bands at expected height.

Samples 2 and 3 are successful.

09.04.2013

Transformation

Investigator: Christian

Aim: Construction of BioBricks → Transformation of mutagenised pJet1.2 iBB2 into E. coli.

Transformation of the PCR-product of iBB2 into E. coli DH5α (30 min ice, 60 sec 42°C, 60 sec ice, 45 min 37°C + 1 ml LB), plated on LB_amp, overnight at 37°C.

Sequencing

Investigator: Christian

Aim: Construction of BioBricks → Sequencing results of iBB3 and iBB4.

IBB3 and iBB4: point mutations correct.

PCR

Investigator: Christian

Aim: Construction of BioBricks → Adding of pre- and suffix via PCR.

PCR with iBB3 and iBB4 (pre- and suffix).

Volume	Reagent	Temp (°C)	Time
1 µl	Phusion polymerase	95	3 min
1 µl	Primer fw	95	30 sec
1 µl	Primer rv	58	45 sec	x19
1 µl	Template	72	4 min
1 µl	dNTPs	72	6 min
10 µl	5x buffer	4	Hold
35 µl	bidest. H2O

PCR

Investigator: Christian

Aim: Construction of BioBricks

The relevant fragment is cut from the gel and then purified via “Qiagen Gel Extraction Kit” (Qiagen, Düsseldorf). Elution into 20 µl elution buffer.

Gel electrophoresis

Gel substances

1% Agarose gel
10 µl RedSafe in 50 ml gel
6 µl GeneRuler™ 2-log DNA Ladder (NEB)
6x loading buffer (for samples)

Lane	Content	Expectations
2	Mutagenesis of iBB2	717 bp
3	Mutagenesis of iBB7	768 bp
4	Mutagenesis of iBB8	1561 bp
5	Digest of pSB1C3-J04450	2000 bp

Band 3 shows a band at the expected heigt, band 4 lies to low, whereas the other two samples are hard to evaluate.

Mutagenesis of iBB7 (band 3) is positive.

PCR

Investigator: Christian

Aim: Construction of BioBricks → Repeat of adding pre- and suffix of iBB1 via PCR.

PCR of iBB1 repeated like 08.04.13.

Transformation

Investigator: Christian

Aim: Preparation of pSB1C3 → Transformation of pSB1C3 J04450 into E. coli.

Transformation of pSB1C3 J04450 into E. coli DH5α (30 min ice, 60 sec 42°C, 60 sec ice, 60 min 37°C + 1 ml LB), plated on LB_CM, overnight at 37°C.

10.04.2013

PCR

Investigator: Christian

Aim: Construction of BioBricks → Cleaning of PCR-products of iBB4.

The relevant fragments were cut from the gel and then purified via “Qiagen Gel Extraction Kit” (Qiagen, Düsseldorf). Elution into 20 µl elution buffer.

Inoculation

Investigator: Christian

Aim: Construction of BioBricks and preparation of pSB1C3 → Inoculation of transformants.

Some colonies of pJet1.2 iBB2 inoculated in 5 ml LB_amp and pSB1C3 J04450 inoculated in 5 ml LB_CM, overnight at 37°C.

PCR

Investigator: Christian

Aim: Construction of BioBricks → Adding of pre- and suffix of iBB5 via PCR.

Volume	Reagent	Temp (°C)	Time
1 µl	Phusion polymerase	95	3 min
1,5 µl	Primer 15 (10 pmol/µl)	95	30 sec
1,5 µl	Primer 16 (10 pmol/µl)	58	45 sec	x30
1 µl	Template (pPha-NR)	72	4 min
1 µl	dNTPs	72	6 min
10 µl	5x buffer	4	Hold
35 µl	bidest. H2O

11.04.2013

Miniprep

Investigator: Christian

Aim: Construction of BioBricks and preparation of pSB1C3, → Miniprep of transformants.

Miniprep of pJet1.2 iBB2 and pSB1C3 J04450 (QIAprep Spin Miniprep Kit (Qiagen)), eluted in 25 µl bidest. H2O.

Digest

Investigator: Christian

Aim: Preparation of BioBrick-templates → Control digestion of pJet1.2 iBB2.

3 µl DNA template
1 µl EcoRI
1 µl PstI
2 µl buffer O (Fermentas)
13 µl bidest. H2O

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

Expectations

pJet1.2: 2430 bp
iBB2: 740 bp

Gel electrophoresis

Gel substances

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

Lane		Content		Expectations
2-7		pJet1.2 + iBB2		2430 + 740 bp

We receive all expected fragments.

All positive.

Digest

Investigator: Christian

Aim: Preparation of pSB1C3 → Digestion with EcoRI and PstI.

50 µl DNA template
2 µl EcoRI
4 µl PstI
8 µl buffer O
6 µl bidest. H2O

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

12.04.2013

Digest

Investigator: Christian

Aim: Construction of BioBricks → Digestion with EcoRI and PstI.

Digestion of iBB1, iBB2, iBB4, iBB5, iBB7 and iBB8.

30 µl DNA template
1 µl EcoRI
1 µl PstI
3,5 µl buffer O

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

PCR

Investigator: Christian

Aim: Construction of BioBricks → Adding of pre- and suffix of iBB3 and iBB6 via PCR.

Volume	Reagent	Temp (°C)	Time
1 µl	Phusion polymerase	95	3 min
1 µl	Primer fw	95	30 sec
1 µl	Primer rv	58	45 sec	x30
1 µl	Template	72	4 min
1 µl	dNTPs	72	6 min
10 µl	buffer	4	Hold
35 µl	bidest. H2O

16.04.2013

Ligation

Investigator: Christian

Aim: Preparation of pSB1C3 → Testligation.

Dephosphorylation of pSB1C3 and testligation with iBB5.

Transformation

Investigator: Christian

Aim: Preparation of pSB1C3 → Transformation of pSB1C3 iBB5 into E. coli.

Transformation of pSB1C3 iBB5 into E. coli DH5α (30 min ice, 60 sec 42°C, 60 sec ice, 60 min 37°C + 1 ml LB), plated on LB_CM, overnight at 37°C.

Digest

Investigator: Christian

Aim: Construction of BioBricks → Digestion with EcoRI and PstI.

Digestion of iBB3 and iBB6.

Inoculation

Investigator: Christian

Aim: Preparation of pSB1C3 → Inoculation of pSB1C3 J04450 for miniprep.

Inoculation of pSB1C3 J04450 in LB_CM, overnight at 37°C.

17.04.2013

Miniprep

Investigator: Christian

Aim: Preparation of pSB1C3 → Miniprep of pSB1C3 J04450.

Miniprep of the liquid cultures (QIAprep Spin Miniprep Kit (Qiagen)), eluted in 25 µl bidest. H2O.

Digest

Investigator: Christian

Aim: Preparation of pSB1C3 → Digestion of pSB1C3 J04450 with EcoRI and PstI.

29 µl DNA template
1 µl EcoRI
1 µl PstI
4 µl buffer O
3 µl bidest. H2O

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

The relevant fragments were cut from the gel and then purified via “Qiagen Gel Extraction Kit” (Qiagen, Düsseldorf). Elution into 20 µl elution buffer.

Gel electrophoresis

Gel substances

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

Lane		Content
2-3		pSB1C3-J04450 cut with EcoRI + PstI.

The plasmid is cut successfully.

Both positive.

Ligation

Investigator: Christian

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

Ligation of iBB1, iBB3, iBB4, iBB5, iBB6, iBB7 and iBB8 into pSB1C3.

18.04.2013

Ligation

Investigator: Christian

Aim: Preparation of pSB1C3 → Self-ligation test.

Ligation of empty pSB1C3.

19.04.2013

PCR

Investigator: Christian

Aim: Construction of BioBricks → Repeat of adding pre- and suffix of iBB8 via PCR.

Reason: Transformation of iBB8 failed, PCR of iBB8 repeated.

Volume	Reagent	Temp (°C)	Time
1 µl	Phusion polymerase	95	3 min
1 µl	Primer fw	95	30 sec
1 µl	Primer rv	65	45 sec	x19
1 µl	Template	72	4 min
1 µl	dNTPs	72	6 min
5 µl	10x buffer	4	Hold
10 µl	bidest. H2O

Gel electrophoresis

Gel substances

1% Agarose gel
10 µl RedSafe in 50 ml gel
6 µl GeneRuler™ 2-log DNA Ladder (NEB)
6x loading buffer (for samples)

Lane		Content		Expectations
3-6		iBB8		1404 bp

We receive all expected fragments. The expected fragment in lane 2 is missing.

The relevant fragments were cut from the gel and then purified via “Qiagen Gel Extraction Kit” (Qiagen, Düsseldorf). Elution into 20 µl elution buffer.

22.04.2013

Miniprep

Investigator: Christian

Aim: Construction of BioBricks → Miniprep of single bricks in pSB1C3.

Miniprep of iBB1, iBB3, iBB4, iBB5, iBB6 and iBB7 (QIAprep Spin Miniprep Kit (Qiagen)), eluted in 25 µl bidest. H2O.

Digest

Investigator: Christian

Aim: Construction of BioBricks → Control digestion of single BioBricks in pSB1C3

Control digestion of iBB1, iBB3, iBB4, iBB5, iBB6 and iBB7.

3 µl DNA template
1 µl EcoRI
1 µl PstI
2 µl buffer O (Fermentas)
13 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 GeneRuler™ 1kb plus DNA Ladder (Thermo Scientific)
6x loading buffer (for samples)

Lane	Content	Expectations
2	pSB1C3-iBB1 cut with EcoRI + PstI	375 bp + 2000 bp
3	pSB1C3-iBB3 cut with EcoRI + PstI	193 bp + 2000 bp
4	pSB1C3-iBB4 cut with EcoRI + PstI	422 + 2000 bp
5	pSB1C3-iBB5 cut with EcoRI + PstI	241 + 2000 bp
6	pSB1C3-iBB6 cut with EcoRI + PstI	273 + 2000 bp
7	pSB1C3-iBB7 cut with EcoRI + PstI	711 + 2000 bp

We receive expected fragments in samples 3 and 4.

2 out of 6 are successful.

Digest

Investigator: Christian

Aim: Construction of BioBricks → Digestion of iBB2 and iBB8.

29 µl DNA template
1 µl EcoRI
1 µl PstI
4 µl buffer O
3 µl bidest. H2O

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

The relevant fragments were cut from the gel and then purified via “Qiagen Gel Extraction Kit” (Qiagen, Düsseldorf). Elution into 20 µl elution buffer.

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		pSB1C3-iBB2		660 bp
4-7		pSB1C3-iBB8		1404 bp

We receive expected fragments for sample 2 and 3.

2 out of 6 are positive.

Ligation

Investigator: Christian

Aim: Construction of BioBricks → Ligation of iBB2 and iBB8 into pSB1C3.

Ligation of iBB2 and iBB8 into pSB1C3, overnight at 16 °C.

23.04.2013

Transformation

Investigator: Christian

Aim: Construction of BioBricks → Transformation pSB1C3 iBB2 and pSB1C3 iBB8 into E. coli.

Transformation of pSB1C3 iBB2 and pSB1C3 iBB8 into E. coli DH5α (30 min ice, 60 sec 42°C, 60 sec ice, 60 min 37°C + 1 ml LB), plated on LB_CM, overnight at 37°C.

Sequencing

Investigator: Christian

Aim: Construction of BioBricks → Sequencing.

PSB1C3 iBB1, iBB3, iBB4, iBB5, iBB6 and iBB7 sent for sequencing.

25.04.2013

Inoculation

Investigator: Christian

Aim: Construction of BioBricks → Inoculation of pSB1C3 iBB2 and pSB1C3 iBB8 for miniprep.

2 colonies of pSB1C3 iBB2 and iBB8 inoculated in 5 ml LB_CM, overnight at 37 °C.

26.04.2013

Sequencing

Investigator: Franzi

Aim: Construction of BioBricks → Examination of sequencing results (23.4.13).

1C3 iBB1 K1 + K2 correct

1C3 iBB4 K1 + K2 correct

1C3 iBB7 K3 + K4 correct

1C3 iBB5 K1 + K2 Mutagenesis of XhoI-Site failed, only necessary for iGEM-standard 10 → unnecessary, rest correct

1C3 iBB3 K1 + K2 same point-Mutation → template?

1C3 iBB6 K1 + K2 same point-mutation → template?

Sequencing

Investigator: Franzi

Aim: Construction of BioBricks → Sequencing of pPhaNR.

Original pPhaNR was sent to sequencing using primers iMR25/26 for iBB3 and iBB6.

Miniprep

Investigator: Franzi

Aim: Construction of BioBricks → Miniprep.

PSB1C3 iBB8 K1 and pSB1C3 iBB2 K1-4 were isolated via Miniprep (QIAprep Spin Miniprep Kit (Qiagen)), eluted in 40 l Elutionbuffer.

Digest

Investigator: Franzi

Aim: Construction of BioBricks → Control digestion of pSB1C3 iBB8 and iBB2.

1 µl DNA template
0,5 µl EcoRI
0,5 µl PstI
1 µl Cut Smart buffer
7 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 GeneRuler™ 2-log DNA Ladder (Thermo Scientific)
6x loading buffer (for samples)

Lane		Content		Expectations
2		pSB1C3-iBB8		1404 bp
3-6		pSB1C3-iBB2		660 bp

IBB8 negative, iBB2 all positive.

Sequencing

Investigator: Franzi

Aim: Construction of BioBricks → Sequencing of pSB1C3 iBB2.

IBB2 K2 + K4 sequenced with primer VF2_fwd and VR_rev.

29.04.2013

PCR

Investigator: Franzi

Aim: Construction of BioBricks → Adding of pre- and suffix of iBB8 via PCR.

Volume	Reagent	Temp (°C)	Time
1 µl	Phusion-Polymerase	95	3 min
1 µl	Primer 33 fw	95	30 sec
1 µl	Primer 34 rv	65	40 sec	x30
1 µl	2xNR template	72	1 min
1 µl	dNTPs	72	7 min
2 µl	DMSO	4	Hold
10 µl	5xGC buffer
33 µl	bidest. H2O

Gel electrophoresis

Gel substances

1% Agarose gel
10 µl RedSafe in 50 ml gel
6 µl GeneRuler™ 2-log DNA Ladder (NEB)
6x loading buffer (for samples)

Lane		Content		Expectations
2-3		iBB8, PCR I		1404 bp
5-6		iBB8, PCR II		1404 bp

The relevant fragments were cut from the gel and then purified via “Qiagen Gel Extraction Kit” (Qiagen, Düsseldorf). Elution into 30 µl bidest. H2O.

All positive.

Digest

Investigator: Franzi

Aim: Construction of BioBricks → Digestion of iBB8

30 µl DNA template
1 µl EcoRI-HF
1 µl PstI-HF
3,6 µl Cut Smart buffer

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

Gel electrophoresis

Ligation

Investigator: Franzi

Aim: Contruction of BioBricks → Assemble the Biobrick iBB8 into the vector pSB1C3.

30 ng vector DNA (pSB1C3; EcoRI/PstI-cut)
270 ng insert DNA (iBB8)
2 µl 10x T4 DNA ligase buffer
1 µl T4 DNA ligase
ad 20 µl bidest. H2O

The samples were incubated overnight at 16 °C.

30.04.2013

Transformation

Investigator: Franzi

Aim: Contruction of BioBricks → Transformation of the plasmid pSB1C3 iBB8 in E. coli.

The complete ligation sample was mixed with one aliquot of chemo-competent E. coli DH5α cells (50 µl) and incubated for 30 min on ice. The heatshock was performed at 42 °C for 60 sec and cells were then incubated at 37 °C for 1 h with 900 µl LB. The sample was concentrated and plated on LB containing chloramphenicol.
The plate was incubated for two days at 28 °C .

Transformation

Investigator: Franzi

Aim: Contruction of BioBricks → Transformation of 2xNR HC+LC.

2xNR HC + LC retransformed as transcribed above. Plated on LB containing ampicillin.

Sequencing

Investigator: Franzi

Aim: Construction of BioBricks → sequencing results.

Sequencing of iBB2 K2 + K4 failed (possibly secondary structure).

Sequenced again with primer cat_int_rev.

Team:Marburg/Notebook:April

From 2013.igem.org

Notebook: April