Daniel:Notebook/ComboLock/2017-1-23
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Circularization Test[edit]
Since the results from the last experiment seemed to indicate that RCA hasn't been working for me, I'm going to try RCA again using the old positive amplicon systems. For examples see Oct-5-2016 and Sept-30-2016. I'll be using PCAmp4
Sample Matrix[edit]
Tube No | Molecules Available | Amount |
0 (AB) | 1.00E+11 | 1 pmol |
2 (AB) | 1.00E+09 | 10 fmol |
4 (AB) | 1.00E+07 | 100 amol |
6 (AB) | 1.00E+05 | 1 amol |
Protocol[edit]
- Latch and Padlock Hybridization
- Suspend 2 uL (8 ug) beads per sample in 100 uL wash buffer
- Apply magnet for 30 sec and remove supernatant
- Add 2 uL oligo per sample to bead solution; incubate at RT for 5 min
- Apply magnet for 30 sec and remove supernatant
- Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant
- Prep 8.2X Padlock0501 mixture buffer
- 16.4 uL 10 uM Padlock0501
- 16.4 uL 10X Amp Ligase Buffer
- 131.2 uL nfH2O
- Heat probes to 95C for 5 minutes
- Chill on ice for 3 minutes
- Add sample to prepared strep beads; vortex to suspend
- Incubate for 30 min at 37C with vigorous shaking
- Circularization
- Prepare 8.5X uL Phusion mix
- Add 20 uL phusion mix to 20 uL reaction
- Incubate at 55C for 4 hours
Reagent | Stock Conc | Final Amount | 1x Vol (uL) | MM Vol (8.5x) (uL) |
NAD+ | 5 mM | 40 nmol | 8 | 68 |
dNTP | 1 mM | 600 pmol | 0.6 | 5.1 |
Betaine | 5 M | 15 umol | 3 | 25.5 |
10X AmpLigase Buffer | 10X | 1X | 2 | 17 |
Amp Ligase | 5 U/uL | 10 U | 2 | 17 |
Phusion HF DNA Polymerase | 2000 U/mL | 6.4U | 3.2 | 27.2 |
nf H2O | 1.2 | 8.4 | ||
Total | 20 | 140 |
Continued tomorrow