IGEM:Groningen/Notebook/iGEM 2011/2011/09/12

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12-9-11



Working on the presentation for Human practices. Made a few calls.

Discussing what to do this week: I am in charge of building the constructs in the chloramphenicol vector, so we can send our
new parts to the parts registry.
This means that I have to clone approximately 30 samples

First a PCR of the plasmid backbone pSB1C3 at 13:45 afterwards: clean up and use 50ng for digestion
Per sample: (2 samples with annealing temperatures of 61,63 and 65 degrees.

Pfu 10× buffer: 5μl
dNTPs 10mM: 1μl
Forward primer: 1μl
Reverse primer: 1μl
pfu DNA polymerase: 1μl
Template: 0.5μl
MQ water: 40.5μl

PCR conditions:
Preheated lid: 111°C
Denaturation: 95°C for 3 min.
Cycle 32×:
denaturation: 95°C for 30s.
annealing: 61,63 and 65°C for 30s.
Extension: 72°C for 4 min.
Final extension: 72°C for 10 min.
Store infinite at 4°C
Finished at 17h, put samples on gel and purify the PCR products.

Making scheme for digestion and ligation of the 31 samples:

Digestion:
Digestion will be done in one reaction (so insert and vector will be in one tube, so don't use fastAP!)
Scheme:
1μl pSB1C3 (50 ng)
6μl plasmid sample
1μl EcoRI
1μl PstI
2μl FD buffer
9μl MQ water

incubate samples for 1h at 37 degrees

Clean up of the digested samples with the High Pure PCR Purification Kit
NOTE: elute in 17 μl MQ water! So it can be used for ligation immediately

Ligation:
17μl digested purified sample
1μl T4 DNA ligase
2μl T4 DNA ligase buffer

Ligate overnight at 4 °C

Purification of the plasmid backbone PCR product after finished PCR at 17:15.
Analysed at 1% agarose gel:
Digestion and ligation was not done, since the PCR of the plasmid backbone failed.
New PCR was done overnight at 19h:

Pfu 10× buffer: 5μl
dNTPs 10mM: 1μl
Forward primer: 1μl
Reverse primer: 1μl
pfu DNA polymerase: 1μl
Template: 0.5μl*
MQ water: 40.5μl
* Template DNA was diluted 1:100

PCR conditions:
Preheated lid: 111°C
Denaturation: 95°C for 3 min.
Cycle 33×:
denaturation: 95°C for 1min.
annealing: 55,60 and 65°C for 30s.
Extension: 72°C for 6 min.
Final extension: 72°C for 10 min.
Store infinite at 4°C



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