User:Robwarden/Notebook/Caspase-3 Activatable Probe/2009/09/08

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PCR Validation

Agarose Gel Purification

  • 1% Agarose
  • Run at 110V for 30 min
  • Band appeared in expected location ~500bp


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.

Gel Purification

  • Ran on 1% agarose at 110V for 25 min.
  • Strong bands seen & removed for each digestion.
  • Purified with qiagen kit.
    • Concentration determined by running 1μL against a ladder.
      • No DNA visible for insert -> undetectable concentration

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

  • Cell Lines:
    • 1X -> Top10
    • 5X -> Stbl2
  • Shook at 30°C for 1:30.
  • Plated on LB-Amp.
  • Incubated overnight at 37°C (1X) and 30°C (5X)


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