User:Anugraha Raman/Notebook/iGEM 2010/2010/06/25

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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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