Daniel:Notebook/ComboLock/2016-11-4

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Revision as of 17:10, 4 November 2016 by >Djacobse (→‎qPCR Sample Table)
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Antibody Binding Test (Started Yesterday)

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Protocol

  1. qPCR
    1. Set up reaction according to table below
    2. Make master mix according to following recipe
      1. uL nfH2O
      2. 252.5 uL SYBR Fast
      3. 25.5 uL 10 uM AmpF
    3. Add 47 uL master mix to each well
    4. qPCR Cycles
      1. 95C 3 min
      2. 95C 3 sec
      3. 55C 30 sec
      4. 72C 20 sec
      5. plate read
      6. goto b x24
      7. 72C 2 min
      8. 16C hold
  2. TBE Gel
    1. Mix 240 uL TBE, 60 uL 6x loading dye
    2. Aliquot 10 uL per sample/ladder lane onto parafilm
    3. Add 2 uL of sample or ladder to correct drop
    4. Load 10 uL in to well
    5. Run gel for 22 minutes at 250V
    6. Open gel and stain with 3 uL SYBR Gold for 3 minutes
    7. Rinse gel and image in gel doc

qPCR Sample Table

Sample Condition Lane AmpR Index Sample Vol (uL) 2X Kapa SYBR qPCR MM 10 uM Forward Primer 10 uM Reverse Primer H2O Total Volume (uL)
Sample 1A 2 ug BSA A1 20 2 25 1 1 21 50
Sample 1B 2 ug BSA A2 20 2 25 1 1 21 50
Sample 2A No Template A3 21 2 25 1 1 21 50
Sample 2B No Template A4 21 2 25 1 1 21 50
Sample 3A No Antibody A5 22 2 25 1 1 21 50
Sample 3B No Antibody A6 22 2 25 1 1 21 50
Sample 4A 20 ng BSA A7 23 2 25 1 1 21 50
Sample 4B 20 ng BSA A8 23 2 25 1 1 21 50
Sample 5A 200 pg BSA H1 24 2 25 1 1 21 50
Sample 5B 200 pg BSA H2 24 2 25 1 1 21 50
NTC qPCR NTC H3 25 0 25 1 1 23 50