Daniel:Notebook/ComboLock/2017-2-28
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RCA + Circularization Test (Started Yesterday)[edit]
Protocol-Part 3[edit]
- ssQubit
- Mix together 796 uL Qubit ssDNA buffer and 4 uL dye (200:1 ratio)
- Add 190 uL mix and 10 uL S1 or S2 to standard tubes
- Add 199 uL mix and 1 uL sample to sample tubes
- Incubate 5 minutes in the dark
- Measure in Qubit; use new run and calibrate with standards
- Results: Both too low to mean anything.
- Column Purification-Qiaquick
- Add 250 uL (5X) PB (binding buffer) to 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 40 uL EB
- Let stand 1 minute
- Spin for 1 minute at 14000 rpm
- Rolling Circle Amplification
- Prepare master mixes with following enzyme layout following the table in this section
- Add 11.5 uL master mix to each tube
- Add 2.5 uL LLRC-01 (10 uM) primer to A and C samples; add 2.5 uL LLRC* (10 uM) primer to B samples
- Add 5 uL sample to new reaction tubes according to following table
- Incubate for 5 minutes at 75C to denature sticky strands
- Lower temperature and incubate for 30 minutes at 55C
- Lower temperature to 30C; allow at least 1 minute of 30C incubation
- Add 1 uL correct Phi29 polymerase and incubate 3 hours at 30C
- Heat kill enzyme with 10 minutes at 65C
Samples | Condition |
Sample X (1-2,ABC) | NEB |
Sample Y (1-2,ABC) | Thermo Fisher |
Sample Z (1-2,ABC) | Epicenter |
A Samples (1-2, XYZ) | Normal Primer |
B Samples (1-2, XYZ) | Phosphorothiorate Primer |
C Samples (1-2, XYZ) | Column Purified Product |
Reagent | Single Rxn Vol | MM-X (NEB) uL | MM-Y (TF) uL | MM-Z (Epi) uL |
Template | 5 | 0 | 0 | 0 |
RCA Primer (10 uM) | 2.5 | 15.25 | 15.25 | 15.25 |
dNTP (1 mM) | 5 | 30.5 | 30.5 | 30.5 |
10X Buffer | 2 | 12.2 | 12.2 | 12.2 |
Phi29 | 1 | 0 | 0 | 0 |
BSA (10 mg/mL) | 0.4 | 2.44 | 0 | 0 |
nfH2O | 4.1 | 25.01 | 27.45 | 27.45 |
Total | 20 | 85.4 | 85.4 | 85.4 |
Protocol-Part 4[edit]
- qPCR
- Make 25.2X qPCR master mix according to following recipe
- 529.2 uL nfH2O
- 630 uL SYBR Fast
- 25.2 uL 10 uM AmpF
- 25.2 uL 10 uM AmpR-Ind20
- Add 48 uL master mix to each well
- Add 2 uL sample according to PCR Plate Layout File:PlateLayout-20170228-RCATest.png
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x40
- 72C 2 min
- 16C hold
- TBE Gel
- Mix 240 uL TBE, 60 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
Results[edit]
- 20170228-QPCR-RCATest.png
qPCR curves
- 2017-02-28-RCATest-NEB.png
Gel image-NEB RCA
- 2017-02-28-RCATest-Gel1.png
Gel image 1 (PreRCA+TF Sample)
- 2017-02-28-RCATest-Gel2.png
Gel image 2 (Epicentre +TF No PO4)