Daniel:Notebook/ComboLock/2017-3-16
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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
- Padlock Hybridization-Y Samples
- Bake samples at 95 C and vortex to resuspend
- Make the following master mix
- 4.2 uL Padlock0601 (10 uM)
- 8.4 uL Amp Ligase 10X buffer
- 50.4 uL nfH2O
- Add 15 uL master mix to appropriate tubes
- Add 5 uL sample according to yesterday's tables
- Incubate at 50C for 20 minutes
- Circularization
- Make the following master mix
- Follow the workflow in yesterday's diagrams
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 |
******
Reagent | Stock Conc | Final Amount | 1x Vol (uL) | MM Vol (4.5x) (uL) |
NAD+ | 5 mM | 40 nmol | 8 | 36 |
dNTP | 1 mM | 600 pmol | 0.6 | 2.7 |
Betaine | 5 M | 15 umol | 3 | 13.5 |
10X AmpLigase Buffer | 10X | 1X | 2 | 9 |
Amp Ligase | 5 U/uL | 10 U | 2 | 9 |
Phusion HF DNA Polymerase | 2000 U/mL | 6.4U | 3.2 | 14.4 |
nf H2O | 1.2 | 5.4 | ||
Total | 20 | 90 |