User:Karmella Haynes/Notebook/BioBrick cloning/2011/01/24

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01/24/11

  • ✓ Minipreps: SP1AB w.t. (6), KAH160, 165, 170/pSB31 (6 each)
  • ✓ Mutagenesis: site directed mutagenesis for SP1AB w.t.
  • ✓ Cultures: KAH160 (10); 2 mL



Minipreps
> SP1AB w.t.: X/P
> Others: X/NcoI (BioBrick constructs contain 2 NcoI sites, vector has 1)

Reagent Volume Expected (approx.):
1-6. SP1AB w.t.
fwd = 3200, 580, 450, 300, 250
rev = 3650, 580, 300, 250

7-12. KAH160/pSB31
fwd = 636, 500+?
rev = 636, 500+?
13-18. KAH165/pSB31
fwd = 636, 344+?
rev = 636, 344+?
19-24. KAH170/pSB31
fwd = 444, 500+?
rev = 444, 500+?
Empty vector
XbaI = 4000, 650, 250, 200
NcoI should generate an extra band
1/24/11 Miniprep digest
15 μL/lane; 1% agarose

1/24/11 Miniprep digest
NcoI only
DNA(plasmid) 2.0 μL
10X buffer 1.5
enzyme 1 1.0
enzyme 2 1.0
dH2O 9.5
  15 μL --> 37°C/ ~15 min.

--> Conclusions: pSB31 does not have an NcoI site. NcoI should generate a short internal band in the BioBrick clones. Only KAH170 worked. Pick more colonies for KAH160 and 165 (10 each).



Site-directed Mutagenesis
> SP1AB has 3 PstI sites
> Stratagene Quick Change mutagenesis kit: Try all three primers at once
--> Forward primers: mut_SP1A 1, mut_SP1B 1, mut_SP1B 2

> Template is ~ 5kb

Reagent Volume  
DNA (plasmid) 1.0 (~100 ng)
10x buffer 2.5
Quick Solution 0.5
10 μM primer 1 1.0
10 μM primer 2 1.0
10 μM primer 3 1.0
dNTP mix 1.0
Quick Change enzyme mix 1.0
dH2O 16.0
  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/ ∞


(1/25/11)
> DpnI Digest (gets rid of methylated template DNA)

  1. Forward strand triple mutation
  2. 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

01/26/11
--> Results: Great ratio (one colony on neg ctrl.). Pick several and check by restriction digest.



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