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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