IGEM:metu/2009/Notebook/wound dressing/2009/09/09

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09.09.2009

1. PCR was done.

Preparation of master mix. 
11 plasmids + 1 control group
10x tag buffer ---- 30 microliter
2 mM dNTP      ---- 30 microliter
25 mM MgCl2    ---- 30 microliter
5 mM t-primer  ---- 30    "
5 mM r-primer  ---- 30    "
1 u/µl tag Enz ---- 12    "
2,5 u/µl pfu   ---- 0.3   "
water nuclease free --- 185.7 µl
aliquot as 49 µl and 1 µl DNA was add.
preparation of dNTP
dATP -- 10 µl
dTTP -- 10 µl
dGTP -- 10 µl
dCTP -- 10 µl
water -- 460 µl
1- initial denaturation - 95 degree centigrade -- 3 minutes
2- denaturation - 95 degree centigrade -- 30 seconds
3- Annealing - 54 degree centigrade -- 30 seconds
4- Extension ( elongation step)  - 72 degree centigrade -- 45 seconds
5- Final elongation - 72 degree centigrade -- 10 minutes (to ensure that any remaining single-stranded DNA is fully extended)

2.PCR purification (clean-up) was done.

3. Electrophorese of PCR products