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

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03/22/11

  • ✓ 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
Reagent SP1AB mut2/3 p65 w.t.
DNA (plasmid, ~100 ng)) 1.0 0.5
10x buffer 2.5 2.5
Quick Solution 0.5 0.5
10 μM primer 1 1.0 1.0
dNTP mix 1.0 1.0
Quick Change enzyme mix 1.0 1.0
dH2O 18.0 18.5
  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/ ∞


(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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