Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/04/30: Difference between revisions

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| ✓ Insert 1 (DBN002)||40 ng ( µL)
| Insert 1 (DBN002)||40 ng ( µL)
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| ✓ Insert 2 (DBN003)||100 ng ( µL)
| Insert 2 (DBN003)||100 ng ( µL)
|-
|-
| ✓ 10x FD Buffer (clear) ||2
| 10x FD Buffer (clear) ||2
|-
|-
| ✓ 10mM DTT||1
| 10mM DTT||1
|-
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| ✓ 10mM ATP||1
| 10mM ATP||1
|-
|-
| ✓ FD BsaI||1
| FD BsaI||1
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|-
| ✓ T4 DNA ligase||0.5
| T4 DNA ligase||0.5
|-
|-
| ✓ M.B. H2O||
| M.B. H2O||
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|-
| Total||20
| Total||20

Revision as of 10:17, 30 April 2015

Ligation/Amplification of DBN002 & DBN003 <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
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Overview

Going to try Type IIS Digestion/Ligation of DBN002 & DBN003, followed by PCR amplification.

Type IIS Digestion/Ligation

Reagent Volume (µL)
Insert 1 (DBN002) 40 ng ( µL)
Insert 2 (DBN003) 100 ng ( µL)
10x FD Buffer (clear) 2
10mM DTT 1
10mM ATP 1
FD BsaI 1
T4 DNA ligase 0.5
M.B. H2O
Total 20

Incubate the ligation reaction in a thermocycler:

  1. 37°C 5 min
  2. 23°C 5 min
  3. Cycle the previous two steps for 6 cycles (total run time 1h)
  4. 4°C hold until ready to proceed

Clean-up of IIS Ligation

Use the Qiagen kit.

PCR

Reagent Volume (uL)
2x master mix 25
MB grade H2O 0
DBN003 f1 primer 0.25
DBN002 r1 primer 0.25
template 24.5
Total 50
Step Temperature °C Duration
Initial denature 95 120 sec
Main Cycle (x40):
denature 95 30 sec
anneal 59 30 sec
extension 72 120 sec
final extension 72 300 sec
soak 4 indef.


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