Team:Marburg/Notebook:April

From 2013.igem.org

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Revision as of 18:27, 3 October 2013

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 were 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 LBamp, oN 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 ddH20  

Gel electrophoresis
gel-electrophoresis-image

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 LBamp, oN 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, Düsseldorf)), eluted in 30 µl 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 (10pmol/µl)   95 30 sec
1,5 µl Primer 14 (10pmol/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 ddH20  
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 (10pmol/µl)   95 30 sec
1,5 µl Primer 16 (10pmol/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 ddH20  

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 LBamp, oN 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 ddH20  
Digest
Investigator: Christian
Aim: Preparation of pSB1C3 → Digestion of pSB1C3 J04450.

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

Gel electrophoresis
gel-electrophoresis-image

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 LBamp, oN 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 ddH20  

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-electrophoresis-image

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 LBCM, oN 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 LBamp and pSB1C3 J04450 inoculated in 5 ml LBCM, oN 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 (10pmol/µl)   95 30 sec
1,5 µl Primer 16 (10pmol/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 ddH20  

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, Düsseldorf)), eluted in 25 µl 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 H2O

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

Expectations

  • pJet1.2: 2430 bp
  • iBB2: 740 bp

Gel electrophoresis
gel-electrophoresis-image

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 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 ddH20  

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 LBCM, oN 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 LBCM, oN 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, Düsseldorf)), eluted in 25 µl 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 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-electrophoresis-image

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 ddH20  

Gel electrophoresis
gel-electrophoresis-image

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, Düsseldorf)), eluted in 25 µl 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 H2O

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

Gel electrophoresis
gel-electrophoresis-image

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 H2O

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

Gel electrophoresis
gel-electrophoresis-image

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)

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

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

Ligation of iBB2 and iBB8 into pSB1C3, oN 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 LBCM, oN 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 LBCM, oN 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, Düsseldorf)), 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 H2O

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

Gel electrophoresis
gel-electrophoresis-image

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)

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 ddH20  

Gel electrophoresis
gel-electrophoresis-image

Gel substances

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

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

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
gel-electrophoresis-image

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)

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

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 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 sec at 42° C for 60 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.