Daniel:Notebook/ComboLock/2017-2-7: Difference between revisions
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Based on [[Daniel:Notebook/ComboLock/2017-2-6|yesterday]]'s conclusions, I've redesigned the experimental protocol to be more sensitive about temperature. The biotin-streptavidin bond is best kept under 70C. I'll use the v3 oligos for now, and try again with v2 if we have a problem. | Based on [[Daniel:Notebook/ComboLock/2017-2-6|yesterday]]'s conclusions, I've redesigned the experimental protocol to be more sensitive about temperature. The biotin-streptavidin bond is best kept under 70C. I'll use the v3 oligos for now, and try again with v2 if we have a problem. | ||
==Sample Matrix== | ==Sample Matrix and Workflow Diagram== | ||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | ||
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'''Workflow Diagram''' | |||
[[Image:20170207-CL-Workflow.png|600px]] | |||
Workflow diagram for the experiment. This experiment tests both the reduced temperatures as well as using ethanol precipitation post-exonuclease digestion. | |||
==Protocol-Part 1== | ==Protocol-Part 1== |
Revision as of 21:12, 8 February 2017
v2 Oligos-Round 3
Based on yesterday's conclusions, I've redesigned the experimental protocol to be more sensitive about temperature. The biotin-streptavidin bond is best kept under 70C. I'll use the v3 oligos for now, and try again with v2 if we have a problem.
Sample Matrix and Workflow Diagram
Sample | Condition |
Sample 1 (AB) | Normal |
Sample 2 (AB) | No Template |
Sample 3 (AB) | No C probes |
Sample 4 (AB) | No phosphate activation |
Workflow Diagram
Workflow diagram for the experiment. This experiment tests both the reduced temperatures as well as using ethanol precipitation post-exonuclease digestion.
Protocol-Part 1
- 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
- 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
Reagent | uL per Sample | uL Total (Samples+NT) | uL Total (No Phosphate) | uL Total (No C Probe) |
PCCA-03 (10 uM) | 1 | 4 | 2 | 0 |
PCCB-03 (10 uM) | 1 | 4 | 2*** | 0 |
Wash buffer | 3 | 12 | 6 | 10 |
Total | 5 | 20 | 10 | 10 |
Protocol-Phase 2
- Lock and Latch Hybridization/Ligation
- Set up the following master mix
- Add 19 uL probe mix to each sample
- Incubate at 55C for 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 the following 8.1X master mix (1X)
- 16.2 uL 10 uM padlock0501(2uL)
- 16.2 uL Amp Ligase 10X reaction buffer(2uL)
- 129.6 uL nfH2O (16uL)
- 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 55C for 2 hours
Reagent | Stock Conc | Final Conc./Amt | uL added | Master Mix (8.2X) |
T4 Ligase Reaction Buffer | 5X | 1X | 4 | 32.8 |
Latch Oligo | 10 uM | 10 pmol | 1 | 8.2 |
Lock Oligo | 10 uM | 10 pmol | 1 | 8.2 |
nfH2O | NA | NA | 13 | 106.6 |
Total | 19 | 155.8 |
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 |
Protocol-Part3
- Exonuclease Digestion
- Mix 15 uL Exo I (20U/uL) and 3 uL ExoIII (100U/uL)
- Add 2 uL to each sample, mix by swirling pipette tip
- Incubate for 1 hour at 37C
- Heat kill by incubating for 5 min at 95C
- Rolling Circle Amplification
- Take 10 uL and set aside for future testing
- Prepare 8.2X Master mix according to table below
- Add 15 uL master mix to each tube
- Add sample to new reaction tubes according to following table
- Incubate at 30C for 6 hours
- Incubate at 65C for 10 minutes;Hold at 10C until next step
- Ethanol Precipitation
- Add 70 uL H2O
- Transfer to a 1.5 mL tube
- Add 300 uL 100% EtOH, 30 uL 3M NaAOc, 1 uL glycoblue
- Incubate overnight at -80C
Reagent | uL Added | Master Mix (8.2X) |
Template | 3 | 0 |
RCA Primer (10 uM) | 2.5 | 20.5 |
dNTP (1 mM) | 0.8 | 6.56 |
10X Buffer | 2 | 16.4 |
Phi29 | 1 | 0 |
nfH2O | 10.7 | 87.74 |
Total | 20 | 131.2 |
Continued tomorrow