Daniel:Notebook/ComboLock/2016-7-22: Difference between revisions
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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. | 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''' | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#C4D79B;font-size:12pt;font-weight:bold" align="center" | |||
| width="110" height="42" | Reagent | |||
| width="65" | Stock | |||
| width="65" | Final | |||
| width="65" | Dilution | |||
| width="74" | Amt in 1 mL (uL) | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | PBS | |||
| align="center" valign="bottom" | 10X | |||
| align="center" valign="bottom" | 1X | |||
| align="center" align="center" valign="bottom" | 10 | |||
| align="center" valign="bottom" | 100 uL | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | SSC | |||
| align="center" valign="bottom" | 20X | |||
| align="center" valign="bottom" | 4X | |||
| align="center" align="center" valign="bottom" | 5 | |||
| align="center" valign="bottom" | 200 uL | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | Rnasin | |||
| align="center" valign="bottom" | 40000 U/mL | |||
| align="center" valign="bottom" | 40 U/mL | |||
| align="center" align="center" valign="bottom" | 100 | |||
| align="center" valign="bottom" | 10 uL | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | nf H2O | |||
| align="center" valign="bottom" | NA | |||
| align="center" valign="bottom" | NA | |||
| align="center" valign="bottom" | NA | |||
| align="center" valign="bottom" | 690 uL | |||
|} | |||
'''LPH Buffer''' | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#C4D79B;font-size:12pt;font-weight:bold" align="center" | |||
| width="65" height="30" | Reagent | |||
| width="105" | Stock | |||
| width="65" | Final | |||
| width="65" | Dilution | |||
| width="65" | Amt in 1 mL (uL) | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | Oligos | |||
| align="center" valign="bottom" | 100 uM | |||
| align="center" valign="bottom" | 100 nM | |||
| align="center" align="center" valign="bottom" | 1000 | |||
| align="center" valign="bottom" | 100 uL | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | SSC | |||
| align="center" valign="bottom" | 20X | |||
| align="center" valign="bottom" | 1X | |||
| align="center" align="center" valign="bottom" | 20 | |||
| align="center" valign="bottom" | 50 uL | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="30" valign="bottom" | Rnasin | |||
| align="center" valign="bottom" | 40000 U/mL | |||
| align="center" valign="bottom" | 40 U/mL | |||
| align="center" align="center" valign="bottom" | 1000 | |||
| align="center" valign="bottom" | 1 uL | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | PBS | |||
| align="center" valign="bottom" | 10X | |||
| align="center" valign="bottom" | 1X | |||
| align="center" align="center" valign="bottom" | 10 | |||
| align="center" valign="bottom" | 100 uL | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="30" valign="bottom" | nf H2O | |||
| align="center" valign="bottom" | NA | |||
| align="center" valign="bottom" | NA | |||
| align="center" valign="bottom" | NA | |||
| align="center" valign="bottom" | 850 uL | |||
|} | |||
==Protocol== | ==Protocol== |
Revision as of 16:57, 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 | 100 | 10 uL |
nf H2O | NA | NA | NA | 690 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
- C-Probe Hybridization
- Measure cell concentrations in cell counter (Human - 400,000 cells/mL)
- Add cells to a new tube such that there are 200,000 cells present (500 uL)
- Pellet cells via centrifugation at 600g for 3 min
- Wash with 200 uL wash buffer and pellet cells via centrifugation at 600g for 3 min
- Add 100 uL 50% hematoxylin (filtered) and pellet cells via centrifugation at 600g for 3 min
- Wash 3 times with 200 uL wash buffer and pellet cells via centrifugation at 600g for 3 min
- Resuspend cells in 100 uL CPH buffer
- Incubate probes at 40C for 1 hour with vigorous agitation
- Wash three times with 200 uL wash buffer and pelleting at 600xg for 3 min
- Incubate cells for 20 min at 40C in 200 uL stringent wash buffer
- Latch and Padlock Hybridization
- Pellet at 600xg for 3 min and remove supernatant
- Resuspend pellet in 100 uL LPH buffer
- Incubate for 30 min at 37C following the sample matrix
- Wash twice with 100 uL wash buffer
- Wash by pelleting cells at 600xg for 3 min
- Resuspend pellet in 100 uL LPH buffer
- Incubate for 30 min at 37C following the sample matrix
- Wash twice with 100 uL wash buffer pelleting cells at 1000xg for 3 min
- 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.