July 1st, 2010
1. Prepare new reactives.
- Primers Preffix FWD and Suffix REV, 200ul at concentration 5uM.
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.
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