User:Karmella Haynes/Notebook/BioBrick cloning/2010/01/30

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01/30/10

  • ✓ Minipreps: KAH139/V0201
  • ✓ Site directed mutagenesis: EF-1α promoter

Minipreps
> Check with XbaI digest

Reagent Volume Expected:Vector = 4500
1-4. KAH139/V0201 = 3655
XbaI digest 01/30/10
15 μL/lane; 1% agarose
DNA(plasmid) 2.0 μL
10X buffer 1.5
XbaI 1.0
dH2O 10.5
  15 μL --> 37°C/ ~15 min.

Site-directed Mutagenesis
> EF-1α promoter contains 2 PstI sites. For quick-change mutagenesis same-strand primers are used to generate single-stranded plasmids for transformation of E. coli. (This actually works???)
> Stratagene Quick Change mutagenesis kit: Try forward and reverse strand mutagenesis (alhtough one is sufficient), since I have both sets of primers on hand...

  1. Forward primers: EF-1a Pst1f, EF-1a Pst2f
  2. Reverse primers: EF-1a Pst1r, EF-1a Pst2r

> Template is ~ 6kb

Reagent Volume  
DNA (plasmid) 0.3 (~100 ng)
10x buffer 2.5
Quick Solution 0.5
10 μM primer 1 1.0
10 μM primer 2 1.0
dNTP mix 1.0
Quick Change enzyme mix 1.0
dH2O 17.7
  25 μL

--> BioRad PCR (Block A)

  • 95°C/ 1 min.
  • [95°C/ 1 min., 55°C/ 1 min., 65°C/ 12 min (2 min./kb)] x30
  • 65°C/ 1 min.
  • 4°C/ ∞


> DpnI Digest (gets rid of methylated template DNA)

  1. Forward strand double mutation
  2. Reverse strand double mutation
  3. 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

02/02/10
--> Ratios look great. ~10 colonies on #1 & 2; 0 colonies on #3 (neg. ctrl)


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