Daniel:Notebook/ComboLock/2016-10-31
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Circularization Optimization Testing
There are several potential difficulties going on right now, but I think the circularization step is the weakest in the protocol. I'm going to test several different methods of circularization:
- T4 polymerase vs Klentaq; according to NEB Klentaq may degrade an encountered downstream strand, which would be a problem potentially. T4 and T7 do not have strand displacement, which will be helpful for circularization
- Time; from what I understand less time is fine and it will be a helpful thing sometimes to not do an overnight step
- RCA; the original idea called for RCA on the final circularized product and not just PCR. Doing some RCA first would greatly increase the amount of product available for PCR and would likely therefore reduce the amount of off-target product such as primer dimers.
Sample Matrix
Sample | Polymerase | Incubation Time | RCA Y/N |
Sample 1 (AB) | Klentaq | ON | Y |
Sample 2 (AB) | Klentaq | ON | N |
Sample 3 (AB) | Klentaq | 2 hr | Y |
Sample 4 (AB) | Klentaq | 2 hr | N |
Sample 5 (AB) | Phusion | ON | Y |
Sample 6 (AB) | Phusion | ON | N |
Sample 7 (AB) | Phusion | 2 hr | Y |
Sample 8 (AB) | Phusion | 2 hr | N |
Protocol
- Template-Bead Binding
- Suspend 2 uL (8 ug) beads per sample in 100 uL wash buffer
- Apply magnet for 30 sec and remove supernatant
- Add 2 uL 10 uM template oligo per sample to bead solution; incubate at RT for 5 min
- Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant
- C Probe Hybridization
- Combine 1 uL of each C probe (10 uM stock) and 3 uL bead wash buffer into a 0.2 mL tube
- Heat C probes to 90C for 5 min; Chill probes on ice to quench
- Add 5 uL probe mixture to beads
- Incubate at 40C for 1 hour with agitation
- Wash beads with 100 uL wash buffer; apply magnet and remove supernatant
- Repeat wash step above
- Add 100 uL cold Low Salt Buffer; apply magnet and remove supernatant
- Latch and Padlock Hybridization
- Add 2 uL Latch0001 (10 uM stock) and 2 uL 10 uM Padlock0201 to each sample to a fresh 0.2 mL tube
- Heat at 65C 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 intermittent mixing
- Apply magnet and remove supernatant
- Add 100 uL wash buffer and vortex to suspend beads; apply magnet and remove supernatant
- Repeat wash step above
- Add 100 uL cold Low Salt Buffer and vortex to suspend; apply magnet and remove supernatant
- Resuspend with 20 uL 1x Amp Ligase buffer
- Circularization-Klentaq (KLN; Samples 1-4,AB)
- Prepare 20 uL KLN mix
- Add 2 uL KLN mix to 20 uL of reaction
- Incubate at 55C for 2 hours or overnight-if 2 hours store at 4C overnight
- Circularization-Phusion(Samples 5-8,AB)
- Prepare 8.1X uL Phusion mix
- Add 20 uL KLN mix to 20 uL of reaction
- Incubate at 55C for 2 hours or overnight-if 2 hours store at 4C overnight
Continued tomorrow
Buffers
KLN Mix
Reagent | Stock | uL added |
Amp Ligase Buffer | 10X | 2 |
dNTPs | 100 uM | 2 |
Hemo Klentaq | NA | 4 |
Amp Ligase | 5 U/uL | 2 |
nfH2O | NA | 10 |
Phusion Buffer
Reagent | Stock Conc | Final Amount | 1x Vol (uL) | MM Vol (8.1x) (uL) |
NAD+ | 5 mM | 40 nmol | 8 | 64.8 |
dNTP | 1 mM | 600 pmol | 0.6 | 4.86 |
Betaine | 5 M | 15 umol | 3 | 24.3 |
10X AmpLigase Buffer | 10X | 1X | 2 | 16.2 |
Amp Ligase | 5 U/uL | 10 U | 2 | 16.2 |
Phusion HF DNA Polymerase | 2000 U/mL | 6.4U | 3.2 | 25.92 |
nf H2O | 1.2 | 9.72 | ||
Total | 20 | 162 |