Daniel:Notebook/ComboLock/2017-6-15
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Antibody Detection Test-Positive Control (Started Yesterday)[edit]
Protocol-Part 3[edit]
- Latch and Bolt Hybridization/Ligation
- Heat kill enzyme by incubating at 65C for 10 minutes
- Padlock Hybridization
- Add 20 uL of following 6.2X Master Mix
- 6.2 uL 10 uM Padlock0601
- 12.4 uL 10X Amp Ligase buffer
- 105.4 uL nfH2O
- Incubate at 70C for 5 min
- Incubate at 50C for 15 minutes
- Wash once with wash buffer*Do not wash sample 5
- Resuspend in 20 uL 1X Amp Ligase buffer
- Circularization
- For phusion samples, make the following master mix
- Add 20 uL circularization mix to each sample
- Incubate at 55C for 2 hours
- Incubate at 95C for 5 minutes
- Hold at 16C
- Exonuclease Digestion
- Mix 12.5 uL Exo I (20U/uL) and 2.5 uL ExoIII (100U/uL)
- Add 2 uL to each sample, mix by swirling pipette tip
- Incubate for 2 hours at 37C
- Heat kill by incubating for 5 min at 95C
******
******
Reagent | Stock Conc | Final Amount | 1x Vol (uL) | MM Vol (10.5x) (uL) |
NAD+ | 5 mM | 40 nmol | 8 | 52 |
dNTP | 1 mM | 600 pmol | 0.6 | 3.9 |
Betaine | 5 M | 15 umol | 3 | 19.5 |
10X AmpLigase Buffer | 10X | 1X | 2 | 13 |
Amp Ligase | 5 U/uL | 10 U | 2 | 13 |
Phusion HF DNA Polymerase | 2000 U/mL | 6.4U | 3.2 | 20.8 |
nf H2O | 1.2 | 7.8 | ||
Total | 20 | 130 |
Protocol-Part 4[edit]
- Rolling Circle Amplification
- Prepare master mix according to table below
- Add 15 uL master mix to each tube
- Add 4 uL sample to appropriate tubes
- Heat reactions up to 95C for 5 min
- Cool to 55C and incubate for 15 minutes
- Cool to 30C and add 1 uL enzyme
- Incubate for 3 hours at 30C
- Incubate for 20 minutes at 65C to denature
Reagent | Single Rxn (uL) | Master Mix X (6.2X) |
Enzyme | NA | Thermo |
Sample | 4 | 0 |
p12RC*** (10 uM) | 0.2 | 1.24 |
RCA Primer LLRC*** (10 uM) | 0.2 | 1.24 |
dNTP (10 mM) | 0.5 | 3.1 |
10X Buffer | 2 | 12.4 |
Phi29 | 1 | 0 |
nfH2O | 11.7 | 72.54 |
Total | 20 | 90.52 |