User:Jorge E. Buendia Buendia/Notebook/iGEM UNAM-Genomics-Mexico/2010/09/01

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September 1st, 2010

1. Run gel to verify purified PCR product of BBa_I20260 with the changed RBS and the double restriction (SpeI-XbaI) of this purified PCR product. This double restriction will not work for our purpose because it eliminates the J23101 promoter + RBS + GFP. I should’ve done the restriction only with XbaI.

1Sept2010.JPG

Lanes: 1) Ladder; 2) Pure PCR product of BBa_I20260 with the changed RBS; 3) BBa_I20260 with the changed RBS double restriction (SpeI-XbaI); 4-8) Claudia's samples.


2. PCR to change RBS into BBa_I20260 (pSB3K3 + J23101 promoter + GFP E0040),

  • PCR will be done with Platinum Taq Polymerase.
  • I will make 2 reactions with primers RBS-GFP FWD-J23101 REV (Tubes 1-2).
  • PCR product is expected to be 3670 bp (941 biopart + 2729 primer pSB3K3).
  • Control: Preffix FWD-Suffix REV (Tube 3).
PCR with Platinum Taq DNA polymerase Volume (ul)
10X PCR Buffer minus M -> 5
10mM dNTP mixture -> 1
50mM MgCl2 -> 1.5
Primer mix (10uM each) -> 1
Platinum Taq DNA Pol -> 0.4
Template DNA -> 2
HPLC -> 35.1
Total volume -> 50
  • If primers are separated and in concentration 5uM, use 1ul of each one.
  • Thermocycler program:
1. 95ºC 5 min
2. 35 cycles
  • 95ºC 45 seg
  • 55ºC 45 seg
  • 72ºC 4 min
3. 72ºC 10 min
4. Hold 4ºC