Team:Marburg/Notebook:April

From 2013.igem.org

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<li>30 ng vector DNA (pSB1C3; EcoRI/PstI-cut)</li>
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<li>30 ng vector DNA (pSB1C3; <i>EcoR</i>I/<i>Pst</i>I-cut)</li>
<li>270 ng insert DNA (iBB8)</li>
<li>270 ng insert DNA (iBB8)</li>
<li>2 µl 10x T4 DNA ligase buffer</li>
<li>2 µl 10x T4 DNA ligase buffer</li>

Revision as of 10:55, 1 October 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
  • 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: 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 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: TBA
  • 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: 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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