August 9, 2007
- Ran PCR cleanup on the 8 PCR rxns that worked from yesterday (all except 0.1uL for Buffer#1). Combined all into one sample.
- Restriction digest with EcoR1 and SpeI
- Assumed we maxed out the spin column so 10ug DNA was eluded in 28uL EB Buffer leading to a DNA concentration of ~357ng/uL
Restriction Digest Protocol
1. Added to tubes:
- 2uL DNA (~350 ng/uL)
- 1uL 10x Buffer
- 1uL BSA
- 0.5uL each restriction enzyme
- 5uL dH2O
2. Gingerly flick tube to mix 3. Spin down tubes briefly 4. Incubate at 37C in water bath for 1-4 hrs (1-2 hrs optimal)
- Also doing restriction on P1010 w/ psB1A2 which will be the vector for the following ligation.
- Started incubating at 12:45pm
- Ran P1010 on a gel to isolate it from psB1A2
- Seems the restriction didnt work as planned. Going to grow up P1010 in psB1A2 overnight and miniprep and restrict again in the morning.