Daniel:Notebook/ComboLock/2017-3-16
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Circularization Comparison (T4 Poly/Ligase)
Protocol-Part 4
- Exonuclease Digestion
- Mix 25 uL Exo I (20U/uL) and 5 uL ExoIII (100U/uL)
- Add 2 uL to each sample, mix by swirling pipette tip
- Incubate for 1.5 hours at 37C
- Heat kill by incubating for 5 min at 95C
- qPCR
- Make qPCR master mixes according to following recipe
- Add 48 uL master mix to each well
- Add 2 uL sample according to plate layout File:PlateLayout-20170316-T4Circ-PreRCATest.png
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x30
- 72C 2 min
- 16C hold
- TBE Gel
- Mix 200 uL TBE and 50 uL 6X dye
- Aliquot 10 uL per sample on to parafilm
- Add 2 uL sample or 1.5 uL ladder to aliquots
- Mix and add 10 uL per lane to gel
- Run gel at 230V for 24 minutes
- Add 2 uL SYBR gold and incubate on shaker for 3 minutes
- Rinse and image in gel doc
Reagent | Single Rxn | MMProd (15.2X) | MM+ (C1, 4.2X) |
Primer Pair (fwd/rev) | NA | primer12*/AmpR-Ind21 | primer24/primer4* |
Forward Primer (10 uM) | 1 | 15.2 | 4.2 |
Reverse Primer (10 uM) | 1 | 15.2 | 4.2 |
nfH2O | 21 | 319.2 | 88.2 |
2X Kapa SYBR Fast | 25 | 380 | 105 |
Total | 48 | 729.6 | 201.6 |
Pre-RCA Results
- 20170316-qPCR-T4Circ-PreRCA.png
qPCR curves
- 2017-03-16-T4Circ-PreRCA.png
Gel image
Protocol-Part 3-Y Samples
- Padlock Hybridization-Y Samples
- Bake samples at 95 C and vortex to resuspend
- Make the following master mix
- 4.2 uL Padlock0601 (10 uM)
- 8.4 uL Amp Ligase 10X buffer
- 50.4 uL nfH2O
- Add 15 uL master mix to appropriate tubes
- Add 5 uL sample according to yesterday's tables
- Incubate at 50C for 20 minutes
- Circularization
- Make the following master mix
- Follow the workflow in yesterday's diagrams
- Exonuclease Digestion
- Mix 7.5 uL Exo I (20U/uL) and 1.5 uL ExoIII (100U/uL)
- Add 2 uL to each sample, mix by swirling pipette tip
- Incubate for 1.5 hours at 37C
- Heat kill by incubating for 5 min at 95C
******
Reagent | Stock Conc | Final Amount | 1x Vol (uL) | MM Vol (4.5x) (uL) |
NAD+ | 5 mM | 40 nmol | 8 | 36 |
dNTP | 1 mM | 600 pmol | 0.6 | 2.7 |
Betaine | 5 M | 15 umol | 3 | 13.5 |
10X AmpLigase Buffer | 10X | 1X | 2 | 9 |
Amp Ligase | 5 U/uL | 10 U | 2 | 9 |
Phusion HF DNA Polymerase | 2000 U/mL | 6.4U | 3.2 | 14.4 |
nf H2O | 1.2 | 5.4 | ||
Total | 20 | 90 |
******
Protocol-Part 5
- Column Purification-Qiaquick-Do this for 20 uL from Y and Z samples
- Add 100 uL (5X) PB (binding buffer) to 20 uL sample
- Load sample onto column and spin for 1 minute at 14000 rpm; discard flow through
- Add 750 uL PE (wash buffer) to column and spin for 1 minute at 14000 rpm; discard flow through;
- Repeat wash step;
- Dry spin column for 1 minute at 14000 rpm; discard flow through
- Let stand with cover open in fume hood for ~5 minutes
- Transfer column to a new 1.5 mL eppendorf tube
- Elute with 30 uL EB
- Let stand 1 minute
- Spin for 1 minute at 14000 rpm
- Rolling Circle Amplification Sample Conversion Table
- Prepare 8.2X Master mix according to table below
- Add 12.5 uL master mix to each tube
- Add 4 uL template according to table
- Add 2.5 uL correct primer according to table
- Incubate at 95 C for 5 minutes
- Lower to 55C for 15 minutes
- Lower to 30C for at least 2 minutes
- Add 1 uL Phi29 polymerase to each sample
- Incubate 3 hours at 30C
- Heat kill enzyme by incubating 10 minutes at 65C; Hold at 12C
Sample Origin | Sample New Letter |
Y | P |
Z | Q |
******
Sample Origin | Sample New Letter |
X | F |
Y | G |
Z | H |
P | I |
Q | K |
Reagent | Single Rxn Vol | 13.2X (A) MM uL | 13.2X (B) MM uL |
Template | 4 | 0 | 0 |
RCA Primer (10 uM) | 2.5 | 33 | 0 |
RCA Phosphothiorate Primer (10 uM) | 2.5 | 0 | 33 |
dNTP (1 mM) | 5 | 66 | 66 |
10X Buffer | 2 | 26.4 | 26.4 |
Phi29 | 1 | 13.2 | 13.2 |
BSA (10 mg/mL) | 0.4 | 5.28 | 5.28 |
nfH2O | 5.1 | 67.32 | 67.32 |
Total | 20 | 211.2 | 211.2 |