Daniel:Notebook/ComboLock/2016-10-22

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Revision as of 20:42, 22 October 2016 by >Djacobse (→‎Time Point qPCRs)
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Cell Test 3 (Started Yesterday)

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Protocol

  1. Circularization
    1. Heat inactivate enzyme by incubating for 2 minutes at 94C
  2. Exonuclease Digestion
    1. Prepare exonuclease I/III mix by mixing 7.5 uL exonuclease I (20 units/μL) and 1.5 uL exonuclease III (100 units/μL) in 1:1 ratio
    2. Add 2 μL of exonuclease I/III mix to reaction
    3. Mix the reaction by swirling pipette around the well 5 times
    4. Incubate reaction at 37 ºC for 2 hours
    5. Heat inactivate enzyme by incubating at 94C for 5 minutes
  3. qPCR
    1. Prepare the following master mix
      1. 89.25 uL nfH2O
      2. 2.55 uL 100 uM AmpF6.4Sol
      3. 127.5 uL Kapa SYBR Fast MM
    2. Add 43 uL master mix to each well
    3. Add sample and AmpR indexes according to sample table below
    4. Sample Condition Lane AmpR Index Sample Vol (uL) 2X Kapa SYBR qPCR MM 100 uM AmpF6.4Sol 10 uM AmpR6.3-IndXX H2O Total Volume (uL)
      Sample 1 Exp. Rep 1 A1 20 2 25 0.5 5 17.5 50
      Sample 2 Exp. Rep 2 B1 20 2 25 0.5 5 17.5 50
      Sample 3 No C Probes C1 21 2 25 0.5 5 17.5 50
      Sample 4 No Cells D1 22 2 25 0.5 5 17.5 50
      NTC No qPCR sample E1 23 0 25 0.5 5 19.5 50
    5. 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
  4. TBE Gel
    1. Mix 56 uL TBE, 14 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

Results


Time Point qPCRs

Testing the 10 uL aliquots I took yesterday that I took at intervals before/after wash/hybridization steps. I'll be using the direct C-Probe amplification primers, and at a lower concentration than usual after yesterday's and Thursday's results.

  1. qPCR
    1. Prepare the following master mixes according to table
    2. Master Mix Forward/Reverse Forward Primer (uL) Reverse Primer (uL) nfH2O (uL) Kapa SYBR Fast MM (uL)
      MM1 p24RC/p4 1.71 1.71 389.88 427.5
      MM2 p6RC/p12 1.71 1.71 389.88 427.5
    3. Add 48 uL master mix to each well
    4. Add 2 uL sample to correct well by following sample table in next section
    5. 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 56 uL TBE, 14 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 Time Point Lane AmpR-F Combo Sample Vol (uL) 2X Kapa SYBR qPCR MM 100 uM Forward Primer 100 uM Reverse Primer H2O Total Volume (uL) Master Mix
Sample 1 Exp. Rep 1 T1 A1 p24RC/p4 2 25 0.1 0.1 22.8 50 MM1
Sample 2 Exp. Rep 2 T1 B1 p24RC/p4 2 25 0.1 0.1 22.8 50 MM1
Sample 3 No C Probes T1 C1 p24RC/p4 2 25 0.1 0.1 22.8 50 MM1
Sample 4 No Cells T1 D1 p24RC/p4 2 25 0.1 0.1 22.8 50 MM1
Sample 1 Exp. Rep 1 T1 E1 p6RC/p12 2 25 0.1 0.1 22.8 50 MM2
Sample 2 Exp. Rep 2 T1 F1 p6RC/p12 2 25 0.1 0.1 22.8 50 MM2
Sample 3 No C Probes T1 G1 p6RC/p12 2 25 0.1 0.1 22.8 50 MM2
Sample 4 No Cells T1 H1 p6RC/p12 2 25 0.1 0.1 22.8 50 MM2
Sample 1 Exp. Rep 1 T2 A3 p24RC/p4 2 25 0.1 0.1 22.8 50 MM1
Sample 2 Exp. Rep 2 T2 B3 p24RC/p4 2 25 0.1 0.1 22.8 50 MM1
Sample 3 No C Probes T2 C3 p24RC/p4 2 25 0.1 0.1 22.8 50 MM1
Sample 4 No Cells T2 D3 p24RC/p4 2 25 0.1 0.1 22.8 50 MM1
Sample 1 Exp. Rep 1 T2 E3 p6RC/p12 2 25 0.1 0.1 22.8 50 MM2
Sample 2 Exp. Rep 2 T2 F3 p6RC/p12 2 25 0.1 0.1 22.8 50 MM2
Sample 3 No C Probes T2 G3 p6RC/p12 2 25 0.1 0.1 22.8 50 MM2
Sample 4 No Cells T2 H3 p6RC/p12 2 25 0.1 0.1 22.8 50 MM2
Sample 1 Exp. Rep 1 T3 A6 p24RC/p4 2 25 0.1 0.1 22.8 50 MM1
Sample 2 Exp. Rep 2 T3 B6 p24RC/p4 2 25 0.1 0.1 22.8 50 MM1
Sample 3 No C Probes T3 C6 p24RC/p4 2 25 0.1 0.1 22.8 50 MM1
Sample 4 No Cells T3 D6 p24RC/p4 2 25 0.1 0.1 22.8 50 MM1
Sample 1 Exp. Rep 1 T3 E6 p6RC/p12 2 25 0.1 0.1 22.8 50 MM2
Sample 2 Exp. Rep 2 T3 F6 p6RC/p12 2 25 0.1 0.1 22.8 50 MM2
Sample 3 No C Probes T3 G6 p6RC/p12 2 25 0.1 0.1 22.8 50 MM2
Sample 4 No Cells T3 H6 p6RC/p12 2 25 0.1 0.1 22.8 50 MM2
Sample 1 Exp. Rep 1 T4 A9 p24RC/p4 2 25 0.1 0.1 22.8 50 MM1
Sample 2 Exp. Rep 2 T4 B9 p24RC/p4 2 25 0.1 0.1 22.8 50 MM1
Sample 3 No C Probes T4 C9 p24RC/p4 2 25 0.1 0.1 22.8 50 MM1
Sample 4 No Cells T4 D9 p24RC/p4 2 25 0.1 0.1 22.8 50 MM1
Sample 1 Exp. Rep 1 T4 E9 p6RC/p12 2 25 0.1 0.1 22.8 50 MM2
Sample 2 Exp. Rep 2 T4 F9 p6RC/p12 2 25 0.1 0.1 22.8 50 MM2
Sample 3 No C Probes T4 G9 p6RC/p12 2 25 0.1 0.1 22.8 50 MM2
Sample 4 No Cells T4 H9 p6RC/p12 2 25 0.1 0.1 22.8 50 MM2
NTC No qPCR sample NA A12 p24RC/p4 0 25 0.1 0.1 24.8 50 MM1
NTC No qPCR sample NA B12 p6RC/p12 0 25 0.1 0.1 24.8 50 MM2