User:Karmella Haynes/Notebook/BioBrick cloning/2010/03/29

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03/29/10

  • ✓ Minipreps: KAH145 (4)
  • ✓ Assembly: KAH86 (re-make, HSVtkTATA-K missing from original)
  • ✓ Site directed mutagenesis (see 01/30/10): DPRE (continue w/ DpnI digest & transformation tomorrow)

Minipreps
> Check with E/P/ScaI digests

Reagent Volume Expected:
Vector = 1784, 1439
1-4. KAH145 = 3555
E/P/ScaI digest 3/29/10
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.

Assembly

  1. KAH86: KAH48/(S/P)/~3280 ✓ + (1) KAH81/(X/P)/2640


> Digests (Fermentas FD)
--> Digest KAH81 w/ ScaI/X/P

Reagent Volume   Cloning digest 3/29/10
30 μL/lane, 1% agarose
DNA (plasmid) up to 24 μL
10x buffer 3.0
enzyme 1 1.0
enzyme 2 1.0
Sca I 1.0
dH2O ---
  30 μL --> 37°C/ ~30 min.


> Measure conc.'s

Sample OD260 260/280 ng/μL
1. KAH81(X/P) 0.215 2.08 10.7

--> KAH48(S/P) = 33.4 ng/μL


> Ligations

Ligation Plate results (lig : neg crtl) 03/30/10
1. KAH81(X/P)/2640, 40 ng + KAH48(S/P)/~3280, 25 ng KAH86 10:1 (Pick 4)
2. KAH48(S/P)/ 25 ng ~50 colonies
  1 2
Insert DNA 4.0 ---
Vector DNA 0.8 0.8
2x lgn buf (Roche) 5.8 5.8
T4 ligase (NEB) 1.0 1.0
dH2O --- 4.0
  11.6 μL 11.6 μL

--> R.T./ ~10 min.; Add 50 μL Z-DH5α


Site-directed Mutagenesis
> Stratagene Quick Change mutagenesis kit; See 01/30/10
> DPRE contains 2 PstI sites.
> Forward primers: mut_DPRE 1, mut_DPRE 2
> Template is ~ 5 kb

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
1 mM 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/ 10 min (2 min./kb)] x30
  • 65°C/ 1 min.
  • 4°C/ ∞


> DpnI Digest of methylated template DNA

  1. Forward strand double mutation
  2. Neg. control (DNA template, 10x buffer, Quick soln. in 25 μL; no PCR)

--> Add 1 μL DpnI enzyme to each sample
--> 37°C/ 1 hr.
--> Transform 50 μL z-DH5α, use 15 μL each sample; Amp plates

03/31/10
--> Ratio: >10:1 (20 Fwd strand mut, 0 neg ctrl); Pick 4


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