User:Jorge E. Buendia Buendia/Notebook/iGEM UNAM-Genomics-Mexico/2010/08/25

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August 25th, 2010

1. Run gel to verify PCR made on August 24th.

Change RBS 25Aug2010.JPG

Lanes: 1) Green ladder; 2) pSB3K3 RBS-GFP FWD-J23101 REV (reaction 1); 3) pSB3K3 RBS-GFP FWD-J23101 REV (reaction 2); 4) Preffix FWD-Suffix REV; 5) Suffix FWD-J23101 REV; 6) RBS-GFP FWD-Suffix REV (Tube 5).

  • This PCR was succesful!! (at least reaction 1)

2. Purify PCR product from pSB3K3 RBS-GFP FWD-J23101 REV (reaction 2) using the High Pure PCR Product Purification Kit Roche.

3. Make restriction to purified PCR product with SpeI-XbaI.

  • Double restriction methods:
DNA -> 5ul
Buffer 2 -> 2ul (10% of total volume)
BSA (required by PstI) -> 1ul
SpeI -> 1ul
XbaI -> 1ul
HPLC -> 10ul (to complete total volume of 20ul)
Incubate at 37ºC 3.5 hrs.

4. Make ligation of BBa_I20260 with the new RBS.

  • Ligation methods (Total volume 20ul):
DNA -> 5ul of p30-MinBP
Buffer for T4 DNA ligase 5X -> 4ul (Final concentration 20%)
T4 DNA ligase -> 1ul
HPLC -> Complete total volume (20ul)
Incubate overnight at 16ºC
Mix well (vortex) buffer and reaction tubes.