Daniel:Notebook/ComboLock/2016-10-21

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Cell Test 3[edit]

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Repeating the cell test but using the qPCR primers as a metric for efficiency throughout the experiment.

Protocol[edit]

  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 CPH buffer according to sample matrix, 1 uL probe mix per sample
    13. Incubate probes at 40C for 1 hour with vigorous agitation
    14. Take 10 uL as for qPCR
    15. Wash three times with 200 uL wash buffer and pelleting at 600xg for 3 min
    16. Incubate cells for 20 min at 40C in 200 uL stringent wash buffer
    17. Take 10 uL as for qPCR
  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. Take 10 uL as for qPCR
    5. Wash twice with 100 uL wash buffer by pelleting cells at 600xg for 3 min
    6. Incubate cells for 20 min at 40C in 150 uL stringent wash buffer
    7. Take 10 uL as for qPCR
    8. 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

    Amplification Primers Test[edit]

    Repeating the experiment from yesterday with a few more conditions. Also, since that gel was saturated I'll be using lower concentrations.

    Gel Image[edit]

    File:2016-10-21-AmplificationPrimers2.png