Daniel:Notebook/ComboLock/2016-10-21

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Revision as of 21:11, 21 October 2016 by >Djacobse (→‎Amplification Primers Test)
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Cell Test 3

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

Protocol

Cell Trial

  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. 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


    Amplification Primers Test

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

    Gel Image

    File:2016-10-21-AmplificationPrimers2.png