User:Robwarden/Notebook/Caspase-3 Activatable Probe/2009/09/11

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PCR Validation

Agarose Gel Purification

  • 1% Agarose
  • Run at 110V for 30 min
  • Band appeared in expected location ~1kb
  • Ladders: NEB 100bp, NEB 1kb


Digestion

 5.0 μL 10X Buffer EcoRI
 0.5 μL 100X BSA
30.0 μL PCR Product / 1 μg Vector (1X and 5X)
 1.0 μL EcoRI
 1.0 μL XhoI
12.5 μL dH2O / to 50 μL
  • Run at 37°C for 1 hr.

Reaction Cleanup

using Qiagen PCR Cleanup Kit

Ligation

2.0 μL 10X T4 Ligase Buffer
2.5 μL Vector Backbone
7.5 μL hgLuc Insert
7.0 μL MQ
1.0 μL T4 DNA Ligase
  • Incubated at RT for 10 min.
  • Killcut with SgrAI for 5 min @ 37°C.

Transformation

  • Stbl2 Cells
  • Shook at 30°C for 1:30.
  • Plated on LB-Amp. (250 μL/plate on 2 plates)
  • Incubated overnight at 30°C

Replating of phgLuc-5x-DNAe

250 μL/plate on 2 plates


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