Friday June 25, 2010
- Digestions w/ purified DNA from yesterday.
- LTP S: 20.4 ng/uL
- LTP A: 12.2 ng/uL
- Bet1 S: 15.4 ng/uL
- Bet1 A: 11.5 ng/uL
- Bet 1.2 S: 9.2 ng/uL
- Bet 1.2 A: 7.7 ng/uL
- Ger 3 S: 11.2 ng/uL
- Ger 3 A: 8.5 ng/uL
- Double Digest (Xba1 & Pst 1):
- 29 uL DNA
- 4 uL 10x NEB Buffer 3
- 4 uL 10x BSA
- .5 uL Xba1
- .5 uL Pst1
- 2 uL H2O
- Total: 40 uL Volume
- Normal Digest (Digested for 1 hour)
- Concentrations after purification
- LTP S: 36.3 ng/uL
- LTP A: 32.1 ng/uL
- Bet1 S: 30 ng/uL
- Bet1 A: 34 ng/uL
- Bet 1.2 S: 32.7 ng/uL
- Bet 1.2 A: 34.3 ng/uL
- Ger 3 S: 11.2 ng/uL
- Ger 3 A: 33.3 ng/uL
- PCR amiRNA
- 1 uL of 164.8 ng/uL DNA template (RS300)
- .5 uL Phusion Polymerase
- 10uL 5x Phusion Buffer
- 1uL 10nm DNTP
- 2.5 uL fwd+rev primer
- 35 uL H20
- Total Volume 50uL reaction
- 3 Reactions
- Reaction 1: Fwd Primer (A) Rev Primer ("primer 4") for LTP/GFP/Betv1
- Reaction 2: Fwd Primer "primer 3" Rev Primer "primer 2" for LTP/GFP/Betv1
- Reaction 3: Fwd Primer "primer 1" Rev Primer (B) for LTP/GFP/Betv1
- Annealing Temp for the three GFP reactions was 67 C and annealing temp for rest of reactions was 60
- Concentrations after purification
- GFP 1; 9.1 ng/uL
- GFP 2: 6.2 ng/uL
- GFP 3: 18.3 ng/uL
- LTP1: 9.9 ng/uL
- LTP2: 7.3 ng/uL
- LTP3: 16.2 ng/uL
- Bet1: 2.6 ng/uL
- Bet2: .5 ng/uL
- Bet3: 3 ng/uL
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