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

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(Autocreate 2009/09/11 Entry for User:Robwarden/Notebook/Caspase-3_Activatable_Probe)
 
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==Entry title==
==PCR Validation==
* Insert content here...
[[Image:090809-hgLuc PCR.jpg|thumb|left]]
===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 dH<sub>2</sub>O / 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)
 




Revision as of 09:34, 11 September 2009

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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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