Daniel:Notebook/PosSequencing/2017-6-1
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Emulsion PCR Test 3 (Started Yesterday)[edit]
Protocol (Continued from Yesterday)[edit]
- Emulsion Breaking
- Set aside 15 uL for qPCR
- With a 200 uL pipette pool the emulsion into a 1.5 mL tube; save the tip and put aside
- Add 100 uL isopropyl alcohol (IPA) to tube with 200 uL tip (use new tip)
- Draw up IPA with tip from part A, mix to resuspend tip
- 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-IPA mix (top layer) but do not disrupt the oil/aqueous interface
- Add 1000 uL IPA and vortex for 30 seconds
- Spin down sample at 13,000rpm for 3 minutes
- Again remove IPA without disturbing the pellet
- Add 500 uL 4X SSC wash buffer to 1.5 mL tube and vortex for 30 seconds
- Go back and mix thoroughly by pipetting up and down; the pellet may be hard to resuspend
- Leave on 1.5mL mixer at 1300rpm 1 minute
- Spin down sample at 13,000rpm for 1 minute
- Remove all but 40uL wash buffer
- Repeat steps 11 to 15 two times (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) and leave on 1.5mL mixer at 1300rpm 1 minute
- 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) and leave on 1.5mL mixer at 1300rpm 1 minute
- Spin down sample at 13,000rpm for 3 minutes
- 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 38 uL master mix to appropriate lanes
- Add 2 uL sample according to plate layout below File:PlateLayout-20170601-SPqPCR-emPCRTest3.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 (9.2X) | Master Mix BioU (9.2X) | Master Mix Dual (5.2X) |
Primer 2 (10 uM) | 1 | 9.2 | 0 | 5.2 |
BioU Primer (10 uM) | 1 | 0 | 9.2 | 5.2 |
2X Kapa SYBR Master Mix | 20 | 184 | 184 | 104 |
nfH2O | 17 | 156.4 | 156.4 | 83.2 |
Total* (+ 2 uL sample=40) | 38 | 349.6 | 349.6 | 197.6 |
Results[edit]
- PlateLayout-20170601-SPqPCR-emPCRTest3.png
Plate layout
- 20170601-SPqPCR-emPCRTest3-plateCTs.png
Plate CT values
- 20170601-SPqPCR-emPCRTest3-normed-plateCTs.png
Plate CT values-normalized
- 20170601-SPqPCR-emPCRTest3-rawcurves.png
Raw curves
Conclusions:
- I need to run this with more cycles; the dual Aqueous and ssDNA showed DNA with exponential amplification but not linear;
- Given the number of cycles for exponential amplification there is not much
- Oil gives no signal (too messy)
- The normalized values show a little better that the only real signal was the dsDNA samples amplified with single primers
Backup on the qPCR log. I'll run this tomorrow