Daniel:Notebook/ComboLock/2016-7-5

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Sequential Hybridization Test

Back to Calendar

Today I'm going to try testing the latch and padlock hybridizations sequentially. This is because of the unusual results of the C probe tests from and 6-30 and July 2.

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 1000 1 uL
nf H2O NA NA NA 700 uL

LPH Buffer

Reagent Stock Final Dilution Amt in 1 mL (uL)
Oligos 100 uM 100 nM 1000 1 uL
SSC 20X 1X 20 50 uL
Rnasin 40000 U/mL 40 U/mL 1000 1 uL
PBS 10X 1X 10 100 uL
nf H2O NA NA NA 850 uL

Experiment

  1. C-Probe Hybridization
    1. Using same cells as 6-28 cells to a new tube such that there are 100000 cells present
      1. Human-256 uL; Mouse-85 uL
    2. Pellet cells via centrifugation at 600g for 3 min
    3. Combine 1.5 uL of each C probe into a 0.2 mL tube
    4. Heat C probes to 90C for 5 min; Chill probes on ice to quench
    5. Add probes to CPH buffer for final concentration of 100 nM (Add 2 uL probe mixture to 0.5mL hybridization buffer)
    6.   C Probes-VIM (C1+C2) C Probes-SOD1 (C1+C2) U87MG 3T3 Cells
      Sample 1 X X X  
      Sample 2     X  
      Sample 3 X X   X
      Sample 4       X
      Sample 5 X X    
    7. Incubate probes at 40C for 1 hour with vigorous agitation
    8. Wash three times with 200 uL wash buffer and pelleting at 600xg for 3 min
    9. 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