Daniel:Notebook/ComboLock/2017-3-16: Difference between revisions
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<li>Exonuclease Digestion</li> | <li>Exonuclease Digestion</li> | ||
<ol type="A"> | <ol type="A"> | ||
<li>Mix | <li>Mix 25 uL Exo I (20U/uL) and 5 uL ExoIII (100U/uL)</li> | ||
<li>Add 2 uL to each sample, mix by swirling pipette tip</li> | <li>Add 2 uL to each sample, mix by swirling pipette tip</li> | ||
<li>Incubate for 1.5 hours at 37C</li> | <li>Incubate for 1.5 hours at 37C</li> |
Revision as of 19:44, 16 March 2017
Circularization Comparison (T4 Poly/Ligase)
Protocol-Part 4
- Exonuclease Digestion
- Mix 25 uL Exo I (20U/uL) and 5 uL ExoIII (100U/uL)
- Add 2 uL to each sample, mix by swirling pipette tip
- Incubate for 1.5 hours at 37C
- Heat kill by incubating for 5 min at 95C
- qPCR
- Make qPCR master mixes according to following recipe
- Add 48 uL master mix to each well
- Add 2 uL sample according to plate layout File:PlateLayout-20170316-T4Circ-PreRCATest.png
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x30
- 72C 2 min
- 16C hold
- Rolling Circle Amplification
- Samples will be aliquoted under following rules
Reagent | Single Rxn | MMProd (15.2X) | MM+ (C1, 4.2X) |
Primer Pair (fwd/rev) | NA | primer12*/AmpR-Ind21 | primer24/primer4* |
Forward Primer (10 uM) | 1 | 15.2 | 4.2 |
Reverse Primer (10 uM) | 1 | 15.2 | 4.2 |
nfH2O | 21 | 319.2 | 88.2 |
2X Kapa SYBR Fast | 25 | 380 | 105 |
Total | 48 | 729.6 | 201.6 |