Daniel:Notebook/ComboLock/2017-3-2
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Padlock0601 Test (Started Yesterday)
Protocol-Part 3
- 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 1.5 hours at 37C
- Heat kill by incubating for 5 min at 95C
- Column Purification-Qiaquick
- Add 150 uL (5X) PB (binding buffer) to 30 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
- Samples will be aliquoted under following rules
- 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
Samples | Condition |
Sample X (1-4, AB) | Unpurified |
Sample Y (1-4, AB) | Purified |
A Samples (1-4) | Normal Primer |
B Samples (1-4) | Phosphorothiorate Primer |
Reagent | Single Rxn Vol | 16.2X MM uL |
Template | 4 | 0 |
RCA Primer (10 uM) | 2.5 | 0 |
dNTP (1 mM) | 5 | 81 |
10X Buffer | 2 | 32.4 |
Phi29 | 1 | 0 |
BSA (10 mg/mL) | 0.4 | 6.48 |
nfH2O | 5.1 | 82.62 |
Total | 20 | 202.5 |
Protocol-Part 4
- BglII Digestion
- Use samples 1 (Experiment) and 4 (No phosphate) for Bglii digest
- Make the following 4.2X master mix
- Aliquot 14 uL master mix into new tubes
- Add 5 uL appropriate sample to each tube
- Use the following thermocycler program
- 5 min 95C
- 10 min 50C
- 1 hr 37C; When this cycle starts add 1 uL Bglii
- Heat kill with 65C for 20 min
- qPCR
- Make 17.2X qPCR master mix according to following recipe
- 361.2 uL nfH2O
- 430 uL SYBR Fast
- 17.2 uL 10 uM AmpF
- Add 47 uL master mix to each well
- Add 2 uL sample and 1 uL appropriate Reverse Primer (1 uM) according to plate layout File:PlateLayout
- 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 192 uL TBE, 48 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
Reagent | Single Rxn Vol | 4.2X MM uL |
RCA Reaction Product | 5 | 0 |
Bglii Cutter Primer (10 uM) | 2 | 8.4 |
10X Buffer 3.1 | 2 | 8.4 |
Bglii | 1 | 0 |
nfH2O | 10 | 42 |
Total | 20 | 58.8 |