User:Karmella Haynes/Notebook/BioBrick cloning/2011/03/22

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


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03/22/11

 * &#x2713; Site directed mutagenesis: p56 and SP1AB mut2/3

Site-directed Mutagenesis > Stratagene Quick Change mutagenesis kit.
 * 1) SP1AB (~5 kb): failed to mutate PstI site #1 last time because of incorrect primer. Ordered new primer mut_SP1A 1
 * 2) p65 (~4 kb): mutate w.t. clone (two PstI sites within single primer); mut_p65 1

--> BioRad PCR (Block A)
 * 95°C/ 1 min.
 * [95°C/ 1 min., 55°C/ 1 min., 65°C/ 10 min (2 min./kb)] x30
 * 65°C/ 1 min.
 * 4°C/ ∞

(3/23/11) > DpnI Digest (gets rid of methylated template DNA)
 * 1) SP1AB mutation
 * 2) SP1AB control (DNA, 10x buffer, Quick soln. in 25 μL; no PCR)
 * 3) p65 mutation
 * 4) p65 control (DNA, 10x buffer, Quick soln. in 25 μL; no PCR)

--> Add 1 μL DpnI enzyme to each sample --> 37°C/ 1 hr. --> Transform 30 μL z-DH5α, use 10 μL each sample; Amp plates

03/24/11 --> Results
 * SP1AB: 4 colonies (0 on neg. ctrl)
 * p65: 2 colonies (0 on neg ctrl.)


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