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.



￼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.


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