Team:Marburg/Notebook:April

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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 cutted with EcoRI and PstI.   2868 bp + 992 bp
3   pPha-T1 cutted 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.

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)

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.

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)
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  

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)

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.

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)
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.

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 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™ 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.

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)
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.

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