Daniel:Notebook/ComboLock/2016-10-18: Difference between revisions

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<li>Combine 0.5 uL of each C probe into a 0.2 mL tube with 2 uL nfH2O</li>
<li>Combine 0.5 uL of each C probe into a 0.2 mL tube with 2 uL nfH2O</li>
<li>Heat C probes to 90C for 5 min; Chill probes on ice to quench</li>
<li>Heat C probes to 90C for 5 min; Chill probes on ice to quench</li>
<li>Add probes to CPH buffer according to sample matrix, 1 uL probe mix per sample</li>
<li>Add probes to 100 uL CPH buffer according to sample matrix, 1 uL probe mix per sample</li>
<li>Incubate probes at 40C for 1 hour with vigorous agitation</li>
<li>Incubate probes at 40C for 1 hour with vigorous agitation</li>
<li>Wash three times with 200 uL wash buffer and pelleting at 600xg for 3 min</li>
<li>Wash three times with 200 uL wash buffer and pelleting at 600xg for 3 min</li>

Revision as of 22:13, 18 October 2016

Cell Trial Padlock0201 (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

Cell Trial 2

There may have been issues with the previous run, so I'm going to repeat as is.

  1. C-Probe Hybridization
    1. Measure cell concentrations in cell counter
    2. Add cells to a new tube such that there are 100,000 cells present per sample
    3.   C Probes-VIM (C1v2+C2) U87MG
      Sample 1 X X
      Sample 2 X X
      Sample 3   X
      Sample 4 X  
    4. Pellet cells via centrifugation at 600g for 3 min
    5. Wash with 200 uL wash buffer and pellet cells via centrifugation at 600g for 3 min
    6. Add 100 uL 50% hematoxylin (filtered) and pellet cells via centrifugation at 600g for 3 min
    7. Wash once with 200 uL wash buffer and pellet cells via centrifugation at 600g for 3 min
    8. Wash 2 times with 200 uL wash buffer prime and pellet cells via centrifugation at 600g for 3 min
    9. Pellet cells via centrifugation at 600g for 3 min
    10. Combine 0.5 uL of each C probe into a 0.2 mL tube with 2 uL nfH2O
    11. Heat C probes to 90C for 5 min; Chill probes on ice to quench
    12. Add probes to 100 uL CPH buffer according to sample matrix, 1 uL probe mix per sample
    13. Incubate probes at 40C for 1 hour with vigorous agitation
    14. Wash three times with 200 uL wash buffer and pelleting at 600xg for 3 min
    15. Incubate cells for 20 min at 40C in 200 uL stringent wash buffer
  2. Latch and Padlock Hybridization
    1. Pellet at 600xg for 3 min and remove supernatant
    2. Resuspend pellet in 100 uL LPH buffer with 100 nM Padlock0201-68 and 100 nM Latch0001
    3. Incubate for 30 min at 37C
    4. Wash twice with 100 uL wash buffer by pelleting cells at 600xg for 3 min
    5. Incubate cells for 20 min at 40C in 150 uL stringent wash buffer
    6. Resuspend with 20 uL 1x Amp Ligase buffer and transfer to a 0.2 mL tube
  3. Circularization
    1. Prepare 20 uL KLN mix
    2. Add 2 uL KLN mix to 20 uL of reaction for sample 1
    3. Incubate at 55C overnight (XX hours)
    4. Continued tomorrow