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

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 * style="background-color: #F2F2F2" align="center"|  |Main project page


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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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