Daniel:Notebook/ComboLock/2016-7-28: Difference between revisions
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<li>Measure cell concentrations in cell counter</li> | <li>Measure cell concentrations in cell counter</li> | ||
<ol type="a"><li>Human - 3.05E5 cells/mL; Mouse - 9.75E5 cells/mL (from [[Daniel:Notebook/ComboLock/2016-7-25|7-25-2016]])</li></ol> | <ol type="a"><li>Human - 3.05E5 cells/mL; Mouse - 9.75E5 cells/mL (from [[Daniel:Notebook/ComboLock/2016-7-25|7-25-2016]])</li></ol> | ||
<li>Add cells to a new tube such that there are | <li>Add cells to a new tube such that there are 30000 cells present per sample</li> | ||
<ol type="a"><li>Human- 350 uL ( | <ol type="a"><li>Human- 350 uL (3 samples) uL; Mouse- 30 uL</li></ol> | ||
<li>Pellet cells via centrifugation at 600g for 3 min</li> | <li>Pellet cells via centrifugation at 600g for 3 min</li> | ||
<li>Wash with 200 uL wash buffer and pellet cells via centrifugation at 600g for 3 min</li> | <li>Wash with 200 uL wash buffer and pellet cells via centrifugation at 600g for 3 min</li> | ||
Line 20: | Line 20: | ||
<li>Wash 2 times with 200 uL wash buffer prime and pellet cells via centrifugation at 600g for 3 min</li> | <li>Wash 2 times with 200 uL wash buffer prime and pellet cells via centrifugation at 600g for 3 min</li> | ||
<li>Pellet cells via centrifugation at 600g for 3 min</li> | <li>Pellet cells via centrifugation at 600g for 3 min</li> | ||
<li>Combine 0.5 uL of each C probe into a 0.2 mL tube with | <li>Combine 0.5 uL of each C probe into a 0.2 mL tube with 2 uL nfH2O</li> | ||
<li>Heat C probes to 90C for 5 min; Chill probes on ice to quench</li> | <li>Heat C probes to 90C for 5 min; Chill probes on ice to quench</li> | ||
<li>Add probes to CPH buffer according to sample matrix, 1 uL probe mix per sample</li> | <li>Add probes to CPH buffer according to sample matrix, 1 uL probe mix per sample</li> | ||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="font-size:12pt" align="center" | |||
| align="center" width="65" height="45" | | |||
|style="background-color:#CCC0DA;font-weight:bold" width="65" | C Probes-VIM (C1+C2) | |||
|style="background-color:#CCC0DA;font-weight:bold" width="65" | C Probes-SOD1 (C1+C2) | |||
|style="background-color:#CCC0DA;font-weight:bold" width="65" | U87MG | |||
|style="background-color:#CCC0DA;font-weight:bold" width="65" | 3T3 Cells | |||
|style="background-color:#CCC0DA;font-weight:bold" width="65" | Treatment | |||
|- style="font-size:12pt" | |||
| height="15" | Sample 1 | |||
| align="center" | X | |||
| align="center" | X | |||
| align="center" | X | |||
| align="center" align="center" | | |||
| align="center" | Klentaq | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| height="15" | Sample 2 | |||
| align="center" | X | |||
| align="center" | X | |||
| align="center" | X | |||
| align="center" align="center" | | |||
| align="center" | Phusion | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Sample 3 | |||
| align="center" align="center" | | |||
| align="center" align="center" | | |||
| align="center" | X | |||
| align="center" align="center" | | |||
| align="center" | Phusion | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| height="15" valign="bottom" | Sample 4 | |||
| align="center" | X | |||
| align="center" | X | |||
| align="center" align="center" | | |||
| align="center" | X | |||
| align="center" valign="bottom" | Phusion | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Sample 5 | |||
| align="center" valign="bottom" | X | |||
| align="center" valign="bottom" | X | |||
| align="center" align="center" valign="bottom" | | |||
| align="center" align="center" valign="bottom" | | |||
| align="center" | Phusion | |||
|} | |||
<li>Incubate probes at 40C for 1 hour with vigorous agitation</li> | <li>Incubate probes at 40C for 1 hour with vigorous agitation</li> | ||
<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> |
Revision as of 16:33, 28 July 2016
Phusion Test
Hemo Klentaq has strand displacement activity, which could be a potential (big) problem for latch-padlock circularization. Therefore, on Chris and Andrew's recommendation I will try a test using Phusion instead of Hemo Klentaq.
Protocol
- C-Probe Hybridization
- Measure cell concentrations in cell counter
- Human - 3.05E5 cells/mL; Mouse - 9.75E5 cells/mL (from 7-25-2016)
- Add cells to a new tube such that there are 30000 cells present per sample
- Human- 350 uL (3 samples) uL; Mouse- 30 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 once with 200 uL wash buffer and pellet cells via centrifugation at 600g for 3 min
- Wash 2 times with 200 uL wash buffer prime and pellet cells via centrifugation at 600g for 3 min
- Pellet cells via centrifugation at 600g for 3 min
- Combine 0.5 uL of each C probe into a 0.2 mL tube with 2 uL nfH2O
- Heat C probes to 90C for 5 min; Chill probes on ice to quench
- Add probes to CPH buffer according to sample matrix, 1 uL probe mix per sample
- 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 with 100 nM Padlock0001-68
- Incubate for 30 min at 37C
- Wash twice with 100 uL wash buffer by pelleting cells at 600xg for 3 min
- Incubate cells for 20 min at 40C in 150 uL stringent wash buffer
- Resuspend pellet in 100 uL LPH buffer with 100 nM Latch0001
- For sample 3, use 10 uL 100 uM Latch0001 (10 uM final concentration)
- Incubate for 30 min at 37C
- Wash twice with 200 uL wash buffer by pelleting cells at 600xg for 3 min
- Incubate cells for 20 min at 40C in 200 uL stringent wash buffer
- Resuspend with 20 uL 1x Amp Ligase buffer and transfer to a 0.2 mL tube
- Circularization
- Prepare 20 uL KLN mix
- Add 2 uL KLN mix to 20 uL of reaction
- Incubate at 55C overnight (XX hours)
- Continued tomorrow
C Probes-VIM (C1+C2) | C Probes-SOD1 (C1+C2) | U87MG | 3T3 Cells | Treatment | |
Sample 1 | X | X | X | Klentaq | |
Sample 2 | X | X | X | Phusion | |
Sample 3 | X | Phusion | |||
Sample 4 | X | X | X | Phusion | |
Sample 5 | X | X | Phusion |