Daniel:Notebook/ComboLock/2016-11-29: Difference between revisions
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Continued [[Daniel:Notebook/ComboLock/2016-11-30|tomorrow]] | Continued [[Daniel:Notebook/ComboLock/2016-11-30|tomorrow]] | ||
[[Category:ComboLock]] [[Category:20161129]] |
Latest revision as of 23:15, 1 December 2016
Extended Latch Test[edit]
The purpose of this experiment is to try and get the circular product. We've had some difficulty with the product containing more of the C-probe (see Nov 18 and Nov 21 results) sequence than they should. So I'm going to try an extended latch that covers the C1 barcode region besides the adapters and UMI. I can give the padlock a 5' phosphate group and then add only Amp ligase. This attach the latch to the padlock at locations where both are present. I can then gap fill.
Sample Matrix[edit]
Sample | Condition |
Sample 1 (AB) | Normal |
Sample 2 (AB) | No Template |
Sample 3 (AB) | No phosphate activation |
Sample 4 (AB) | No C Probe |
Protocol[edit]
- 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; start part 3 after incubation begins
- 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
- Phosphorylation
- Set up reaction according to table
- Incubate at 37C for 30 min
- Heat kill enzyme with 15 minutes at 65C
- Latch and Padlock Hybridization
- Set up the following master mix
- Add 19 uL probe mix to each sample; for sample 3 don't add activated Padlock, add normal padlock0201
- Incubate at 95C for 5 min
- Lower the temperature to 55C
- Without removing the reaction from the incubator add 1 uL Amp Ligase to each reaction
- Incubate at 55C for 2 hours
- Circularization
- Prepare 9X uL Phusion mix
- Add 20 uL Phusion mix to 20 uL of reaction
- Incubate at 60C for 2 hours
Reagent | Stock Conc | Final Conc./Amount | uL added |
AmpLigase Reaction Buffer | 10X | 1X | 2 |
ATP | 10 mM | 1 mM | 2 |
Padlock0201 | 10 uM | 150 pmol total | 15 |
T4 DNA Kinase | 10 U/uL | 10 U | 1 |
nfH2O | NA | NA | 0 |
Total | 20 |
Reagent | Stock Conc | Final Conc./Amount | uL added | Master Mix (6.2X) |
AmpLigase Reaction Buffer | 10X | 1X | 2 | 12.4 |
Latch Oligo | 10 uM | 20 pmol | 2 | 12.4 |
Phosphate Reaction | NA | NA | 2 | 12.4 |
nfH2O | NA | NA | 13 | 80.6 |
Total | 19 | 117.8 |
Reagent | Stock Conc | Final Amount | 1x Vol (uL) | MM Vol (9x) (uL) |
NAD+ | 5 mM | 40 nmol | 8 | 72 |
dNTP | 1 mM | 600 pmol | 0.6 | 5.4 |
Betaine | 5 M | 15 umol | 3 | 27 |
10X AmpLigase Buffer | 10X | 1X | 2 | 18 |
Amp Ligase | 5 U/uL | 10 U | 1 | 9 |
Phusion HF DNA Polymerase | 2000 U/mL | 6.4U | 4.2 | 37.8 |
nf H2O | 1.2 | 10.8 | ||
Total | 20 | 180 |
Continued tomorrow