User:Karmella Haynes/Notebook/BioBrick cloning/2010/07/20

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07/20/10

 * &#x2713; Site-directed mutagenesis: fly mCherry

Site-directed Mutagenesis > fly mCherry contains 1 PstI site > Stratagene Quick Change mutagenesis kit > Forward primer: mut_flymCh 1 > Template is 4930 kb

--> BioRad PCR (Block A) --> Continue later
 * 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/ ∞

07/22/10 > DpnI Digest (gets rid of methylated template DNA) --> Add 1 μL DpnI enzyme to each sample --> 37°C/ 1 hr. (Left reaction digesting for 2 hours instead...very busy day!) --> Transform 50 μL Turbo DH5α, use 10 μL each sample; Amp plates
 * 1) Mutagenesis reaction
 * 2) Control (0.3 μL template DNA, 2.5 μL 10x buffer, 0.5 μL Quick soln., 21.7 μL dH2O; no PCR)

07/23/10 --> Result: 4 colonies on Mutagenesis plate, 0 on neg. ctrl.; Pick all 4 and grow 2 mL cultures for minipreps (see 7/23/20)


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