Daniel:Notebook/ComboLock/2017-1-6: Difference between revisions
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[[Category:ComboLock]] [[Category:20170106]] |
Revision as of 18:09, 9 January 2017
Version 2 Oligos
The version 2 oligos have several changes. The main is that they are shorter and use a UMI on the padlock instead of the latch. I will test these oligos and the new protocol. I'm also going to check
Sample Matrix
Sample | Condition |
Sample 1 (AB) | Normal |
Sample 2 (AB) | No Template |
Sample 3 (AB) | No C probes |
Sample 4 (AB) | No phosphate activation |
Protocol-Phase 1
- Phosphorylation-MirrorC Probe B (PCCB-02)
- Set up reaction according to table
- Incubate at 37C for 30 min
- Heat kill enzyme with 15 minutes at 65C
- 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 following 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 4 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-Lock oligo
- Set up reaction according to table
- Incubate at 37C for 30 min
- Heat kill enzyme with 15 minutes at 65C
- Lock and Latch Hybridization/Ligation
- Set up the following master mix; Note: for sample 4 add un-phosphorylated Lock0201
- 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 temperature to 40C; incubate 20 minutes
- Lower the temperature to 20C; wait 30 seconds then take off incubator
- Add 1 uL T4 Ligase to each reaction
- Incubate at RT for 30 min
- Heat kill enzyme by incubating at 65C for 10 minutes
- Wash twice with 100 uL wash buffer
- Padlock Hybridization
- Make following master mix
- 16.4 uL 10 uM padlock0401
- 16.4 uL Amp Ligase 10X reaction buffer
- 131.2 uL nfH2O
- Resuspend sample in 20 uL padlock buffer
- Incubate at 37C for 30 minutes
- Circularization
- Prepare 8.5X uL Phusion mix
- Add 20 uL phusion mix to 20 uL reaction
- Incubate at 40C for 4 hours
Reagent | Stock Conc | Final Conc./Amount | uL added |
T4 Polynucleotide Kinase Buffer | 10X | 1X | 2 |
ATP | 10 mM | 1 mM | 2 |
PCCB-02 | 10 uM | 100 pmol total | 10 |
T4 DNA Kinase | 10 U/uL | 10 U | 1 |
nfH2O | NA | NA | 5 |
Total | 20 |
Reagent | uL per Sample | uL Total |
PCCA | 1 | 8 |
Mirror C Probe B reaction | 2 | 16 |
Wash buffer | 2 | 16 |
Total | 5 | 40 |
Protocol-Phase 2
Reagent | Stock Conc | Final Conc./Amount | uL added |
T4 DNA Ligase Buffer | 10X | 1X | 2 |
ATP | 10 mM | 1 mM | 2 |
Lock0201 | 10 uM | 10 pmol total | 10 |
T4 DNA Kinase | 10 U/uL | 10 U | 1 |
nfH2O | NA | NA | 5 |
Total | 20 |
Reagent | Stock Conc | Final Conc./Amt | uL added | Master Mix (6.2X) | Master Mix (2.2X) |
T4 Ligase Reaction Buffer | 5X | 1X | 4 | 24.8 | 8.8 |
Latch Oligo | 10 uM | 10 pmol | 1 | 6.2 | 2.2 |
Lock Oligo Phosphate Reaction | (5 uM Lock) | 10 pmol | 2 | 12.4 | *2.2* |
nfH2O | NA | NA | 12 | 86.8 | 39.2 |
Total | 19 | 74.4 | 26.4 |
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
Buffers
Wash Buffer
Reagent | Stock | Final | Dilution | Amt in 20 mL |
NaCl | 1.5 M | 0.5 M | 3 | 6.66 mL |
Tris-HCl | 500 mM | 20 mM | 25 | 800 uL |
EDTA | 0.5M | 1 mM | 500 | 40 uL |
nf H2O | NA | NA | NA | 12.5 mL |