User:Karmella Haynes/Notebook/BioBrick cloning/2010/05/26

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05/26/10

  • ✓ MV3 and MV8 vector construction: transformation (C2925 dam-/dcm-)
  • ✓ MV3 and MV8 vectors construction: PCR hygromycin resistance from V0200



Transformation
> Transform plasmids into C2925 dam-/dcm- cells (50 μL per transf.)

  1. pcDNA3.1+ neo (MV1), 0.5 μL + 10 μL dH2O
  2. pcDNA3.1+ neo ΔpCMV (MV7), 0.5 μL + 10 μL dH2O
  3. dH2O, 10 μL

> Use "quick and dirty" method (heat shock on plate)

> 5/27/10: Lots of colonies on plasmid plates, zero on neg. ctrl. Success! 2 miniprep cultres per plasmid.



PCR
> Amplify hygromycin resistance gene (1023 bp) from V0200
> Primers:

  • Hygro f1 (contains BsaBI site and start codon)
  • Hygro r1 (contains BstBI site)
Reagent Volume  
DNA (plasmid) 0.5
10 μM primer mix 1.0
2x GoTaq green mix 12.5
dH2O 11.0
  25.0

--> BioRad Block A

  • 95°C/ 3 min.
  • [95°C/ 1 min., 57°C/ 1 min., 72°C/ 1 min.] x30
  • 72°C/ 3 min.
  • 4°C/ ∞

--> Zymo clean the PCR; elute w/ 20 μL dH2O (continue on 5/27)


Reagent Volume   HygroR PCR digest 05/27/10
30 μL/lane, 1% agarose
DNA (PCR) 20.0
BsaBI 1.0
BstBI 1.0
NEB buffer 4 3.0
dH2O 6.0

--> 60°C (BsaBI)/ 1 hr.; 65°C (BstBI)/ 1 hr.
--> Gel purify

> Measure conc.

Sample OD260 260/280 ng/μL
1. HygroR (BsaBI/BstBI) 0.899 2.04 44.9



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