Daniel:Notebook/ComboLock/2016-7-22: Difference between revisions

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>Djacobse
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<li>Wash three times with 200 uL wash buffer and pelleting at 600xg for 3 min</li>
<li>Wash three times with 200 uL wash buffer and pelleting at 600xg for 3 min</li>
<li>Incubate cells for 20 min at 40C in 200 uL stringent wash buffer</li>
<li>Incubate cells for 20 min at 40C in 200 uL stringent wash buffer</li>
</ol>
<li>Latch and Padlock Hybridization</li>
<li>Latch and Padlock Hybridization</li>
<ol type="A">
<ol type="A">

Revision as of 20:44, 22 July 2016

Cell Retention Test

I've been worried about cell retention during the spins for some time, and I've been thinking about how to test it. So, I'm going to run through the protocol (without probes) but using hematoxylin to stain the cells.

Buffers

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 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

Protocol

  1. C-Probe Hybridization
    1. Measure cell concentrations in cell counter (Human - 400,000 cells/mL)
    2. Add cells to a new tube such that there are 200,000 cells present (500 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 3 times with 200 uL wash buffer and pellet cells via centrifugation at 600g for 3 min
    7. Resuspend cells in 100 uL CPH buffer
    8. Incubate probes at 40C for 1 hour with vigorous agitation
    9. Wash three times with 100 uL wash buffer and pelleting at 600xg for 3 min
    10. Incubate cells for 20 min at 40C in 150 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
    3. Incubate for 30 min at 37C following the sample matrix
    4. Wash twice with 100 uL wash buffer
      1. Wash by pelleting cells at 600xg for 3 min
    5. Resuspend pellet in 100 uL LPH buffer
    6. Incubate for 30 min at 37C following the sample matrix
    7. Wash twice with 100 uL wash buffer pelleting cells at 1000xg for 3 min
    8. Resuspend with 20 uL 1x Amp Ligase buffer

This is the last step where you change buffer in the protocol. If I still have cells, we're good.

Results

I still had cells at this point, but the hematoxylin really helps visualize the cell pellet and doesn't interfere with the chemistry (so it has been reported) so I think that it is a good idea to use it.

Latch 100 and Dextran Test

Buffer Prep

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

Protocol

  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
    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 3 times with 200 uL wash buffer and pellet cells via centrifugation at 600g for 3 min
      1. Human- uL; Mouse- uL
    7. Pellet cells via centrifugation at 600g for 3 min
    8. Combine 17 uL of each C probe into a 0.2 mL tube
    9. Heat C probes to 90C for 5 min; Chill probes on ice to quench
    10. Add probes to CPH buffer according to sample matrix
    11.   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
    12. Incubate probes at 40C for 1 hour with vigorous agitation
    13. Wash three times with 200 uL wash buffer and pelleting at 600xg for 3 min
    14. 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 200 uL wash buffer by pelleting cells at 600xg for 3 min
    5. Incubate cells for 20 min at 40C in 200 uL stringent wash buffer
    6. Resuspend pellet in 100 uL LPH buffer with 100 nM Latch0001
    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
  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