Daniel:Protocols/qPCR

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qPCR[edit]

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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
  1. Basic thermocycler protocol:
    1. Initial Denaturation (95C, >1 min)
    2. Denaturation (95C, 3-10 sec)
    3. Annealing (50C-60C), 20-40 sec)
    4. Extension (~72C, 15-40 sec)
    5. plate read (reads amt DNA present)
    6. repeat 2-4, (10-30 times)
    7. Final extension (~72C, >1 min)
    8. Cool (kill) reaction, 16C hold

Assorted qPCR Protocols[edit]

High Resolution Chromosome Painting[edit]

  1. 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)
  1. Aliquot 100 uL into each of xxNxx qPCR tubes
  2. PCR protocol:
    1. PCR 3 min 95C
    2. 95C 3 sec
    3. 55C 30 sec
    4. 72C 20 sec
    5. plate read
    6. goto ii x17
    7. 72C 2 min
    8. 16C hold
  3. Pool 3 samples together
  4. Column Purification, elute with 40 uL EB
  5. Measure concentrations in nanodrop


Presets[edit]

  1. "Standard" protocol (used for most things)
    1. 95C 2 min
    2. 95C 20 sec
    3. 58C 30 sec
    4. 70C 30 sec
    5. Goto b X times (Variable, usually about 40)
    6. 4C forever