User:Robwarden/Notebook/Caspase-3 Activatable Probe/2009/09/10: Difference between revisions

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Colonies 2, 5, 7, and 13 were submitted for sequencing.  T7 was used for the forward primer. BHG was used for the reverse primer.  Both primers were supplied by the sequencing facility.
Colonies 2, 5, 7, and 13 were submitted for sequencing.  T7 was used for the forward primer. BHG was used for the reverse primer.  Both primers were supplied by the sequencing facility.


==PCR of hRLuc8==
===Reaction Setup===
  5.0 μL 10X Buffer
  1.0 μL hRLuc8 plasmid
  2.5 μL Primer "hRLuc8_EcoRI_Fwd"
  2.5 μL Primer "hRLuc8_XhoI_Rev"
  1.0 μL 10mM dNTP mix
  0.5 μL Vent Polymerase
37.5 μL dH<sub>2</sub>O
===Thermo Cycle===
{| {{table}}
!Cycles || Temp (°C) || Time
|-
| 1 || 94 || 10:00
|-
| rowspan="3" | 30
| 94 || 0:45
|-
| 50 || 0:30
|-
| 72 || 1:30
|-
| rowspan="2" | 1
| 72 || 10:00
|-
| 4 || ∞
|}


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Revision as of 12:56, 10 September 2009

Caspase-3 Activatable Probe <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

Colony Screening

Miniprep

with Qiagen kit

Digestion

  • 20 μL reaction volume
  • NEB Buffer 4
  • .5 μL of EcoRI
  • .5 μL of SgrAI
  • 10 μL of DNA
  • Incubate at 37°C for 1 hr

Agarose Gel

  • 1% Agarose, run for 30 min at 120V
Gel 1
Lane 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Sample - 1kb 1 2 3 4 5 6 7 8 9 10 100bp
Gel 2
Lane 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Sample - 1kb 11 12 13 14 15 16 17 18 100bp
  • Predicted Banding Patterns
    • Correct: 6535
    • Incorrect: 6297, 1434
Gel Images
Gel 1
Gel 2
  • All colonies appeared to work, except for #12

Sequencing

Colonies 2, 5, 7, and 13 were submitted for sequencing. T7 was used for the forward primer. BHG was used for the reverse primer. Both primers were supplied by the sequencing facility.

PCR of hRLuc8

Reaction Setup

 5.0 μL 10X Buffer
 1.0 μL hRLuc8 plasmid
 2.5 μL Primer "hRLuc8_EcoRI_Fwd"
 2.5 μL Primer "hRLuc8_XhoI_Rev"
 1.0 μL 10mM dNTP mix
 0.5 μL Vent Polymerase
37.5 μL dH2O

Thermo Cycle

Cycles Temp (°C) Time
1 94 10:00
30 94 0:45
50 0:30
72 1:30
1 72 10:00
4 ∞


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