User:Karmella Haynes/Notebook/BioBrick cloning/2011/02/23

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02/23/11

  • ✓ Cultures/ minipreps: G luc (4); 2 mL each (sequence confirm tomorrow)
  • Mutagenesis: activation domains SP1AB 2/3 (mutate PstI site #1); p65 (wait for new primer)

--> mut_SP1A 1 is incorrect; re-order



Minipreps
> Check with E/P digests

Reagent Volume Expected:
1-4. G luc (KAH016) = 558
File:KAH 022311 gel1.tif
15 μL/lane; 1% agarose
DNA(plasmid) 2.0 μL
10X buffer 1.5
EcoRI 1.0
PstI 1.0
dH2O 9.5
  15 μL --> 37°C/ ~15 min.



Site-directed Mutagenesis
> SP1AB: Mutated PstI sites 2 and 3 last time; still need to mutate site #1
> Stratagene Quick Change mutagenesis kit:

  1. SP1AB 2/3, primer mut_SP1A 1
  2. p65, primer mut_p65 1

> Templates are ~ 5kb and 4kb

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/ ∞


(2/24/11)
> DpnI Digest (gets rid of methylated template DNA)

  1. SP1AB 2/3 mut rxn
  2. SP1AB 2/3 control (DNA, 10x buffer, Quick soln. in 25 μL; no PCR)
  3. p65 mut rxn
  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 DH5α Turbo, use 10 μL each sample; Amp plates


2/25/11
--> Results