Team:Marburg/Notebook:April

From 2013.igem.org

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<span class="aim">Aim:</span>
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<span class="aim-desc">TBA Transformation of the plasmid pSB1C3 iBB8 in <i>E. coli</i> DH5.</span>
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<span class="aim-desc">TBA Transformation of the plasmid pSB1C3 iBB8 in <i>E. coli</i> DH5&alpha;.</span>
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Revision as of 19:36, 30 September 2013

Notebook: April

26.04.2013

Sequencing
Investigator: Franzi
Aim: 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: Sequencing of pPhaNR.

Original pPhaNR was sent to sequencing using primers imR25/26 for iBB3 and iBB6.

Miniprep
Investigator: Franzi
Aim: TBA

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: TBA
  • 1 µl DNA template
  • 0,5 µl EcoRI
  • 0,5 µl PstI
  • 1 µl Cut Smart buffer
  • 6x loading 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)

IBB8 negative, iBB2 all positive.
Sequencing
Investigator: Franzi
Aim: Sequencing of pSB1C3 iBB2.

IBB2 K2 + K4 sequenced with primer VF2_fwd and VR_rev.

29.04.2013

PCR
Investigator: Franzi
Aim: TBA
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 x17
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)

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: TBA
  • 30 µl DNA template
  • 1 µl EcoRI-HF
  • 1 µl PstI-HF
  • 3,6 µl Cut Smart buffer
  • 6x loading 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)

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: TBA 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: TBA Transformation of the plasmid pSB1C3 iBB8 in E. coli DH5α.

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: TBA

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

Sequencing
Investigator: Franzi
Aim: TBA

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

Sequenced again with primer cat_int_rev.

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