Daniel:Notebook/ComboLock/2017-2-2
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RCA Time Test (Started Friday Jan 27)[edit]
Phosphorothiorate RCA Primer (Started Yesterday)[edit]
- qPCR
- Make qPCR master mix according to following recipe
- 361.2 uL nfH2O
- 17.2 uL AmpF6.4
- 430 uL Kapa SYBR Fast
- Add 47 uL master mix to each well
- Add 2 uL sample and 1 uL AmpR indexed according to table
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x24
- 72C 2 min
- 16C hold
- TBE Gel
- Mix 94 uL TBE, 24 uL 6x loading dye
- Aliquot 10 uL per sample/ladder lane onto parafilm
- Add 2 uL of sample or ladder to correct drop
- Load 10 uL in to well
- Run gel for 23 minutes at 230V
- Open gel and stain with 3 uL SYBR Gold for 3 minutes
- Rinse gel and image in gel doc
Sample | Sample | Condition | AmpR Index | Lane | Sample Vol (uL) | 2X Kapa SYBR qPCR MM | 10 uM Forward Primer | 10 uM Reverse Primer | H2O | Total Volume (uL) |
Sample 1AN | Sample | 3 hours | 20 | A8 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1BN | Sample | 3 hours | 20 | A7 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3AN | No C Probe | 3 hours | 21 | A6 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3BN | No C Probe | 3 hours | 21 | A5 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1AO | Sample | 3 hours-Epi | 22 | A4 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1BO | Sample | 3 hours-Epi | 22 | A3 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3AO | No C Probe | 3 hours-Epi | 23 | A2 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3BO | No C Probe | 3 hours-Epi | 23 | A1 | 2 | 25 | 1 | 1 | 21 | 50 |
NTC | 24 | H1 | 0 | 25 | 1 | 1 | 23 | 50 |
Results[edit]
- 20170202-qPCR-RCATimeTest-Phosphorothiorate.png
qPCR curve
- 2017-02-02-RCATimeTest-Phosphorothiorate.png
Gel image
Version 2 Oligos Round 2[edit]
Since I've been having so much trouble with the version 3 oligos, I want to test the v2 oligos again.
Sample Matrix[edit]
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[edit]
- 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 20 min
- Heat kill enzyme by incubating at 65C for 10 minutes
- Wash twice with 100 uL wash buffer
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 | 6 |
Mirror C Probe B reaction | 2 | 12 |
Wash buffer | 2 | 12 |
Total | 5 | 30 |
Protocol-Phase 2[edit]
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 |
This table had errors. Correct table below but I used the table above
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 | 74.4 | 28.6 |
Total | 19 | 117.8 | 39.6 |
Protocol-Part 3-Padlock Hybridization and Circularization[edit]
- Padlock Hybridization
- Make the following master mix
- 16.4 uL 10 uM Padlock0401A
- 16.4 uL Amp Ligase 10X reaction buffer
- 131.2 uL nfH2O
- Resuspend sample in 20 uL padlock buffer
- Incubate at 37C for 10 minutes
- Circularization
- Prepare 8.5X uL Phusion mix
- Add 20 uL phusion mix to 20 uL reaction
- Incubate at 40C for 4 hours
- Heat kill enzyme by incubating 10 minutes at 95C
- Hold overnight at 12C
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[edit]
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 |