User:Jorge E. Buendia Buendia/Notebook/iGEM UNAM-Genomics-Mexico/2010/07/01

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July 1st, 2010

1. Prepare new reactives.

  • Primers Preffix FWD and Suffix REV, 200ul at concentration 5uM.

dNTPs 0.4mM.

2. Make PCR to amplify GFP E0240.

  • Tubes were marked 1-2:
  • GFP E0240 new primers, new dNTP`s
  • Ctrl 1 kb new primers, new dNTP’s

3. Run gel to verify PCR to amplify GFP E0240.

PCR GFP E0240 1Jul2010.JPG

Lanes: 1) Ladder; 2) GFP E0240 new, new dNTP`s (Tube 1); 3) Ctrl 1 kb new primers, new dNTP’s (Tube 2).

4. Purify GFP E0240 PCR product using the High Pure PCR Product Purification Kit Roche. Tube marked: “GFP E0240 Purified

5. Make XbaI-PstI double restriction to GFP E0240 extracted by PCR.

Double restriction methods XbaI and PstI (DNA 10 ul, Total volume 30 ul):
DNA -> 10 ul
Buffer 3 -> 3 ul (10% of total volume)
BSA (required by SpeI) -> 1 ul
PstI -> 1.5 ul
XbaI -> 1.5 ul
HPLC -> 13 ul (to complete total volume of 30ul)
Incubate at 37ºC overnight