Daniel:Notebook/ComboLock/2016-7-11: Difference between revisions
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<li>Combine 17 uL of each C probe into a 0.2 mL tube</li> | <li>Combine 17 uL of each C probe into a 0.2 mL tube</li> | ||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | ||
|- style="background-color:#CCC0DA;font-size:12pt" align="center | |- style="background-color:#CCC0DA;font-size:12pt;font-weight:bold" align="center" | ||
| width="104" height=" | | width="104" height="45" | Sample | ||
| width="65" | Cell Type | | width="65" | Cell Type | ||
| width="65" | uL 100 uM C Probe | | width="65" | uL 100 uM C Probe | ||
Line 155: | Line 155: | ||
| align="center" | 1 | | align="center" | 1 | ||
| 100 nM | | 100 nM | ||
| align="center" | 0. | | align="center" | 0.01 | ||
| align="center" | | | align="center" | 6.8 | ||
| align="center" | 2 | | align="center" | 2 | ||
Line 164: | Line 164: | ||
| align="center" | 5 | | align="center" | 5 | ||
| 500 nM | | 500 nM | ||
| align="center" | 0. | | align="center" | 0.05 | ||
| align="center" | | | align="center" | 7.5 | ||
| align="center" | 10 | | align="center" | 10 | ||
Line 173: | Line 173: | ||
| align="center" | 10 | | align="center" | 10 | ||
| 1 uM | | 1 uM | ||
| align="center" | 0. | | align="center" | 0.1 | ||
| align="center" | 8 | | align="center" | 7.8 | ||
| align="center" | 20 | | align="center" | 20 | ||
Line 191: | Line 191: | ||
| align="center" | 1 | | align="center" | 1 | ||
| 100 nM | | 100 nM | ||
| align="center" | 0. | | align="center" | 0.01 | ||
| align="center" | | | align="center" | 6.8 | ||
| align="center" | 2 | | align="center" | 2 | ||
Line 200: | Line 200: | ||
| align="center" | 5 | | align="center" | 5 | ||
| 500 nM | | 500 nM | ||
| align="center" | 0. | | align="center" | 0.05 | ||
| align="center" | | | align="center" | 7.5 | ||
| align="center" | 10 | | align="center" | 10 | ||
Line 209: | Line 209: | ||
| align="center" | 10 | | align="center" | 10 | ||
| 1 uM | | 1 uM | ||
| align="center" | 0. | | align="center" | 0.1 | ||
| align="center" | 8 | | align="center" | 7.8 | ||
| align="center" | 20 | | align="center" | 20 | ||
Line 227: | Line 227: | ||
| align="center" | 1 | | align="center" | 1 | ||
| 100 nM | | 100 nM | ||
| align="center" | 0. | | align="center" | 0.01 | ||
| NA | | NA | ||
| align="center" | 2 | | align="center" | 2 |
Revision as of 18:28, 11 July 2016
Million Fold Test
After conversations with Dr. Zhang and Andrew on Friday I'm going to try a SOD1 test using 5X and 10X amounts from the original 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 | 100 | 10 uL |
nf H2O | NA | NA | NA | 690 uL |
LPH Buffer
Reagent | Stock | Final | Dilution | Amt in 100 uL (uL) |
Oligos | 10 uM | 100 nM | 100 | 1 uL |
SSC | 20X | 1X | 5 | 20 uL |
Rnasin | 40000 U/mL | 40 U/mL | 100 | 1 uL |
PBS | 10X | 1X | 10 | 10 uL |
nf H2O | NA | NA | NA | 68 uL |
KLN Mix
Reagent | Stock | uL added |
Amp Ligase Buffer | 10X | 2 |
dNTPs | 100 uM | 2 |
Hemo Klentaq | NA | 4 |
Amp Ligase | 5 U/uL | 2 |
nfH2O | NA | 10 |
Experiment
- C-Probe Hybridization
- Measure cell concentrations in cell counter
- Human - cells/mL; Mouse - cells/mL
- Add cells to a new tube such that there are 100000 cells present
- Human- uL; Mouse- uL
- Pellet cells via centrifugation at 600g for 3 min
- Combine 17 uL of each C probe into a 0.2 mL tube
- Heat C probes to 90C for 5 min; Chill probes on ice to quench
- Add probes to CPH buffer for final concentration of 500 or 1000 nM (Add 2 uL probe mixture to 0.5mL hybridization 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; use Latch0001 and Padlock0001-68
- Incubate for 30 min at 37C following the sample matrix
- Wash twice with 200 uL wash buffer
- Wash by pelleting cells at 600xg for 3 min
- Resuspend with 20 uL 1x Amp Ligase buffer
- Circularization
- Prepare 20 uL KLN mix
- Add 2 uL KLN mix to 20 uL of reaction
- Incubate at 55C overnight (XX hours)
- Continued tomorrow
Sample | Cell Type | uL 100 uM C Probe | Final C Probe Concentration | Final C Probe Amount (nmol) | Estimated Fold (log10) | uL Mix to add |
Hs-1 | U87MG | 1 | 100 nM | 0.01 | 6.8 | 2 |
Hs-2 | U87MG | 5 | 500 nM | 0.05 | 7.5 | 10 |
Hs-3 | U87MG | 10 | 1 uM | 0.1 | 7.8 | 20 |
Hs-4 | U87MG | 0 | 0 | 0 | NA | 0 |
mm-1 | 3T3 | 1 | 100 nM | 0.01 | 6.8 | 2 |
mm-2 | 3T3 | 5 | 500 nM | 0.05 | 7.5 | 10 |
mm-3 | 3T3 | 10 | 1 uM | 0.1 | 7.8 | 20 |
mm-4 | 3T3 | 0 | 0 | 0 | NA | 0 |
NTC | NA | 1 | 100 nM | 0.01 | NA | 2 |