Daniel:Notebook/PosSequencing/2017-6-20
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Emulsion PCR Test 5 (Started Yesterday)[edit]
Protocol[edit]
- Prepare Water saturated compounds
- Mix 3 mL diethyl ether and 3 mL ddH2O in a falcon tube; allow to settle, extract top (ether) phase
- Mix 3 mL ethyl acetate and 3 mL ddH2O in a falcon tube; allow to settle, extract top (acetate) phase
- Emulsion Breaking
- With a 200 uL pipette add 100 uL diethyl ether to each sample; save the tip
- Adjust tip to 200 and using same tip pool samples in a new 1.5 mL tube
- Vortex the pooled sample in the 1.5 mL tube for 30 seconds
- Spin down the sample 13,000 rpm for 5 minutes
- Take off oil-DE mix (top layer) but do not disrupt the oil/aqueous interface
- Add 1000 uL ethyl acetate and vortex for 30 seconds
- Spin down sample at 13,000rpm for 3 minutes
- Remove ethyl acetate without disturbing the pellet
- Add 1000 uL diethyl ether and vortex for 30 seconds
- Spin down sample at 13,000rpm for 3 minutes
- Remove DE without disturbing the aqueous phase
- Add 500 uL 4X SSC wash buffer to 1.5 mL tube and vortex for 30 seconds
- Spin down sample at 13,000rpm for 1 minute
- Remove all but 40uL wash buffer
- Repeat steps 11 to 15 one more time (j to o)
- On last wash, take 15 uL (of 100) for qPCR
- Strand Separation (DNA Denaturation)
- Add 500 uL KOH buffer (pH 12.0)
- Spin down sample at 13,000rpm for 1 minute
- Remove all but 40uL buffer
- Repeat KOH steps A-C once more
- Add 500 uL 4X SSC buffer (pH 12.0)
- Spin down sample at 13,000rpm for 1 minute
- Remove all but 10uL buffer
- Resuspend in 80 uL 2X SSC buffer
- Use this for final step of qPCR
- Single primer qPCR
- Make the following master mixes
- Aliquot 35 uL master mix to appropriate lanes
- Add 2 uL sample according to plate layout below File:PlateLayout-SinglePrimer-20170620-emPCRTest5.png
- Run the following thermocycler program
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x30
- 72C 2 min
- 16C hold
******
Reagent | uL in Single Rxn | Master Mix P2 (6.2X) | Master Mix BioU (6.2X) | Master Mix Dual (6.2X) |
Primer 2 (10 uM) | 1 | 6.2 | 0 | 6.2 |
BioU Primer (10 uM) | 1 | 0 | 6.2 | 6.2 |
2X Kapa SYBR Master Mix | 20 | 124 | 124 | 124 |
nfH2O | 14 | 86.8 | 86.8 | 80.6 |
Total* (+ 5 uL sample=40) | 35 | 217 | 217 | 217 |
Results[edit]
- PlateLayout-SinglePrimer-20170620-emPCRTest5.png
Plate layout
- EmPCR5-singleprimer-plateCTs-normed.png
Plate CTs-normed
- EmPCR5-singleprimer-rawcurves.png
Raw curves
So the dual primer NTC amplified and none of the sample lanes had (control did well before). This means the single primer lanes will not amplify either, so there was no point in carrying the qPCR any further.