Daniel:Notebook/ComboLock/2016-7-25

From ZhangLabWiki
Jump to navigation Jump to search

Latch 100 and Dextran Test[edit]

Back to Calendar

Buffer Prep[edit]

Stringent Wash Buffer

Reagent Stock Final Dilution Amt in 5 mL (uL)
PBS 10X 1X 10 500 uL
SSC 20X 4X 5 1000 uL
Rnasin 40000 U/mL 40 U/mL 1000 5 uL
nf H2O NA NA NA 4.5 mL

LPH Buffer

Reagent Stock Final Dilution Amt in 1 mL (uL)
Oligos 100 uM 100 nM 1000 100 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

CPH Buffer with 10% Dextran Sulfate

Reagent Stock Conc. Final Conc. Amt. Added
SSC 20X 1X 50 uL
Tween 20 10% 0.10% 10 uL
Rnasin 40000 U/mL 40 U/mL 1 uL
Riboside Vanadyl Complex 200 mM 20 mM 100 uL
Poly Vinylsulfonic Acid 25% 2.50% 100 uL
Salmon Sperm 10 mg/mL 100 ug/mL 10 uL
Dextran Sulfate 25% 10% 400 uL
Probes 100 uM 100 nM 1 uL/probe
Water NA NA 340 uL
Total     1 mL

Wash Buffer Prime (Wash buffer with 2.5% polyvinyl sulfonic acid since it is better at removing free pigment from hematoxylin)

Reagent Stock Final Dilution Amt in 10 mL (uL)
PBS 10X 1X 10 1000 uL
Poly Vinylsulfonic Acid 25% w/v 2.5% w/v 10 1000 uL
Tween 20 10% 0.10% 100 100 uL
Rnasin 40000 U/mL 4 U/mL 10000 1 uL
nf H2O NA NA NA 7.9 mL

Protocol[edit]

  1. C-Probe Hybridization
    1. Measure cell concentrations in cell counter
      1. Human - 3.05E5 cells/mL; Mouse - 9.75E5 cells/mL
    2. Add cells to a new tube such that there are 25000 cells present per sample
      1. Human- 328 uL (4 samples) uL; Mouse- 26 uL
    3. Pellet cells via centrifugation at 600g for 3 min
    4. Wash with 200 uL wash buffer and pellet cells via centrifugation at 600g for 3 min
    5. Add 100 uL 50% hematoxylin (filtered) and pellet cells via centrifugation at 600g for 3 min
    6. Wash once with 200 uL wash buffer and pellet cells via centrifugation at 600g for 3 min
    7. Wash 2 times with 200 uL wash buffer prime and pellet cells via centrifugation at 600g for 3 min
    8. Pellet cells via centrifugation at 600g for 3 min
    9. Combine 0.5 uL of each C probe into a 0.2 mL tube with 3 uL nfH2O
    10. Heat C probes to 90C for 5 min; Chill probes on ice to quench
    11. Add probes to CPH buffer according to sample matrix, 1 uL probe mix per sample
    12.   C Probes-VIM (C1+C2) C Probes-SOD1 (C1+C2) U87MG 3T3 Cells Treatment
      Sample 1 X X X   None
      Sample 2 X X X   Dextran
      Sample 3 X X X   10X Latch
      Sample 4     X   None
      Sample 5 X X   X None
      Sample 6 X X     None
    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 Padlock0001-68
    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 pellet in 100 uL LPH buffer with 100 nM Latch0001
      1. For sample 3, use 10 uL 100 uM Latch0001 (10 uM final concentration)
    7. Incubate for 30 min at 37C
    8. Wash twice with 200 uL wash buffer by pelleting cells at 600xg for 3 min
    9. Incubate cells for 20 min at 40C in 200 uL stringent wash buffer
    10. 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
    3. Incubate at 55C overnight (XX hours)
    4. Continued tomorrow