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

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>Djacobse
>Djacobse
Line 33: Line 33:
| align="center" valign="bottom" | 40000 U/mL
| align="center" valign="bottom" | 40000 U/mL
| align="center" valign="bottom" | 40 U/mL
| align="center" valign="bottom" | 40 U/mL
| align="center" align="center" valign="bottom" | 100
| align="center" align="center" valign="bottom" | 1000
| align="center" valign="bottom" | 10 uL
| align="center" valign="bottom" | 1 uL


|- style="background-color:#D9D9D9;font-size:12pt"
|- style="background-color:#D9D9D9;font-size:12pt"
Line 41: Line 41:
| align="center" valign="bottom" | NA
| align="center" valign="bottom" | NA
| align="center" valign="bottom" | NA
| align="center" valign="bottom" | NA
| align="center" valign="bottom" | 690 uL
| align="center" valign="bottom" | 700 uL


|}
|}

Revision as of 16:58, 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 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
    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.