Daniel:Notebook/PosSequencing/2017-6-2
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Emulsion PCR Test 3 (Started Wednesday)
Protocol-Part 2
- 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-Round2.png
- Run the following thermocycler program
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x150
- 72C 2 min
- 16C hold
Reagent | uL in Single Rxn | Master Mix P2 (5.2X) | Master Mix BioU (5.2X) | Master Mix Dual (5.2X) |
Primer 2 (10 uM) | 1 | 5.2 | 0 | 5.2 |
BioU Primer (10 uM) | 1 | 0 | 5.2 | 5.2 |
2X Kapa SYBR Master Mix | 20 | 104 | 104 | 104 |
nfH2O | 17 | 88.4 | 88.4 | 83.2 |
Total* (+ 2 uL sample=40) | 38 | 197.6 | 197.6 | 197.6 |
Results
- PlateLayout-20170601-SPqPCR-emPCRTest3-Round2.png
Plate layout
- 20170602-SinglePrimer-emPCRTest3-plateCTs.png
Plate CT values
- 20170602-SinglePrimer-emPCRTest3-norm-plateCTs.png
Plate CT values-normalized
- 20170602-SinglePrimer-emPCRTest3-norm-rawcurves.png
Raw curves
Conclusions:
- It didn't show well, but the H4 lane (dsDNA +++ control, dual primer) had a weird curve that started negative and went to zero quickly. It could have been poor baseline correction (platewise instead of by lane or something), in which case it actually CT'd 1st.