Daniel:Notebook/ComboLock/2016-7-11

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Revision as of 18:18, 11 July 2016 by >Djacobse (→‎Buffer Prep)
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Million Fold Test

Back to Calendar

After conversations with Dr. Zhang and Andrew on Friday I'm going to try a SOD1 test using 5X and 10X amounts from the original protocol.

Buffer Prep

Stringent Wash Buffer

Reagent Stock Final Dilution Amt in 1 mL (uL)
PBS 10X 1X 10 100 uL
SSC 20X 4X 5 200 uL
Rnasin 40000 U/mL 40 U/mL 100 10 uL
nf H2O NA NA NA 690 uL

LPH Buffer

Reagent Stock Final Dilution Amt in 100 uL (uL)
Oligos 10 uM 100 nM 100 1 uL
SSC 20X 1X 5 20 uL
Rnasin 40000 U/mL 40 U/mL 100 1 uL
PBS 10X 1X 10 10 uL
nf H2O NA NA NA 68 uL

KLN Mix

Reagent Stock uL added
Amp Ligase Buffer 10X 2
dNTPs 100 uM 2
Hemo Klentaq NA 4
Amp Ligase 5 U/uL 2
nfH2O NA 10

Experiment

  1. C-Probe Hybridization
    1. Measure cell concentrations in cell counter
      1. Human - cells/mL; Mouse - cells/mL
    2. Add cells to a new tube such that there are 100000 cells present
      1. Human- uL; Mouse- uL
    3. Pellet cells via centrifugation at 600g for 3 min
    4. Combine 17 uL of each C probe into a 0.2 mL tube
    5. Sample Cell Type uL 100 uM C Probe Final C Probe Concentration Final C Probe Amount (nmol) Estimated Fold (log10) uL Mix to add
      Hs-1 U87MG 1 100 nM 0.05 7.5 2
      Hs-2 U87MG 5 500 nM 0.25 8.2 10
      Hs-3 U87MG 10 1 uM 0.5 8.5 20
      Hs-4 U87MG 0 0 0 NA 0
      mm-1 3T3 1 100 nM 0.05 7.5 2
      mm-2 3T3 5 500 nM 0.25 8.2 10
      mm-3 3T3 10 1 uM 0.5 8.5 20
      mm-4 3T3 0 0 0 NA 0
      NTC NA 1 100 nM 0.05 NA 2
    6. Heat C probes to 90C for 5 min; Chill probes on ice to quench
    7. Add probes to CPH buffer for final concentration of 500 or 1000 nM (Add 2 uL probe mixture to 0.5mL hybridization buffer)
    8. Incubate probes at 40C for 1 hour with vigorous agitation
    9. Wash three times with 200 uL wash buffer and pelleting at 600xg for 3 min
    10. 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; use Latch0001 and Padlock0001-68
    3. Incubate for 30 min at 37C following the sample matrix
    4. Wash twice with 200 uL wash buffer
      1. Wash by pelleting cells at 600xg for 3 min
    5. Resuspend with 20 uL 1x Amp Ligase buffer
  3. Circularization
    1. Prepare 20 uL KLN mix
    2. Add 2 uL KLN mix to 20 uL of reaction
    3. Incubate at 55C overnight (XX hours)
    4. Continued tomorrow