User:Jorge E. Buendia Buendia/Notebook/iGEM UNAM-Genomics-Mexico/2010/09/02

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September 2nd, 2010

1. Run gel to verify PCR made on September 1st.

Lanes: 1) Green ladder; 2) BBa_I20260 RBS-GFP FWD-J23101 REV (Reaction 1); 3) BBa_I20260 RBS-GFP FWD-J23101 REV (Reaction 2); 4) BBa_I20260 Preffix FWD-Suffix REV; 5-15) Claudia’s samples.


2. Purify PCR product from BBa_I20260 RBS-GFP FWD-J23101 REV (reaction 1 and 2) using the High Pure PCR Product Purification Kit Roche.


3. Make restriction to purified PCR product with XbaI.

  • Restriction methods:
DNA -> 5ul
Buffer 4 -> 2ul (10% of total volume)
BSA -> 1ul
XbaI -> 1ul
HPLC -> 11ul (to complete total volume of 20ul)
  • Incubate at 37ºC 3.5 hrs.


4. Inactivate restriction enzyme, 20 min at 65ºC.


5. Make ligation of BBa_I20260 with the new RBS.

  • Ligation methods (Total volume 20ul):
DNA -> 5ul
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.



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