Daniel:Notebook/ComboLock/2016-6-24: Difference between revisions
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<li>Prepare exonuclease I/III mix by mixing 1 uL exonuclease I (20 units/μL) and 5 uL exonuclease III (100 units/μL) in 1:1 ratio</li> | <li>Prepare exonuclease I/III mix by mixing 1 uL exonuclease I (20 units/μL) and 5 uL exonuclease III (100 units/μL) in 1:1 ratio</li> | ||
<li>Add 2 μL of exonuclease I/III mix to reaction</li> | <li>Add 2 μL of exonuclease I/III mix to reaction</li> | ||
<li>Mix the reaction by swirling pipette around the well 5 times</li> | <li>Mix the reaction by swirling pipette around the well 5 times</li> |
Revision as of 23:29, 22 June 2016
Stage 2 Test (Started yesterday)
Protocol
- Latch and Padlock Hybridization
- Heat inactivate enzyme by incubating for 2 minutes at 94C
- Exonuclease Digestion
- Prepare exonuclease I/III mix by mixing 1 uL exonuclease I (20 units/μL) and 5 uL exonuclease III (100 units/μL) in 1:1 ratio
- Add 2 μL of exonuclease I/III mix to reaction
- Mix the reaction by swirling pipette around the well 5 times
- Incubate reaction at 37 ºC for 2 hours
- Heat inactivate enzyme by incubating at 94C for 5 minutes
- qPCR
- Set up reaction according to table
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x20
- 72C 2 min
- 16C hold