Daniel:Protocols/qPCR
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qPCR[edit]
qPCR is different for every experiment, both in how you make master mixes and cycle number/times/temperatures. However, this page has the basics of qPCR and also template qPCR protocols for my experiments.
Basic qPCR Protocol[edit]
Basic qPCR uses these ingredients:
- DNA Template
- Primers (forward and reverse)
- KAPA SYBRFast Master Mix
- H2O to bring to reaction volume
- Basic thermocycler protocol:
- Initial Denaturation (95C, >1 min)
- Denaturation (95C, 3-10 sec)
- Annealing (50C-60C), 20-40 sec)
- Extension (~72C, 15-40 sec)
- plate read (reads amt DNA present)
- repeat 2-4, (10-30 times)
- Final extension (~72C, >1 min)
- Cool (kill) reaction, 16C hold
Assorted qPCR Protocols[edit]
High Resolution Chromosome Painting[edit]
- Reaction table
10 nM OligoPool | 2X Kapa SYBR qPCR MM | 100 uM AP1V61U | 100 uM AP2V6 | 2 mM aa-dUTP | H20 | Total Volume | |
per rxn | 0.4 | 50 | 0.4 | 0.4 | 2.5 | 46.3 | 100 |
BSPP 1 (xxNxx.1x) |
- Aliquot 100 uL into each of xxNxx qPCR tubes
- PCR protocol:
- PCR 3 min 95C
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto ii x17
- 72C 2 min
- 16C hold
- Pool 3 samples together
- Column Purification, elute with 40 uL EB
- Measure concentrations in nanodrop
Presets[edit]
- "Standard" protocol (used for most things)
- 95C 2 min
- 95C 20 sec
- 58C 30 sec
- 70C 30 sec
- Goto b X times (Variable, usually about 40)
- 4C forever