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

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07/20/10

  • ✓ 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

Reagent Volume  
DNA (plasmid) 0.3 (~100 ng)
10x buffer 2.5
Quick Solution 0.5
10 μM primer 1.0
1 mM dNTP's 1.0
Quick Change enzyme mix 1.0
dH2O 18.7
  25 μL

--> 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/ ∞

--> Continue later


07/22/10
> DpnI Digest (gets rid of methylated template DNA)

  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)

--> 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


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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