Daniel:Notebook/ComboLock/2016-10-21: Difference between revisions
Jump to navigation
Jump to search
>Djacobse |
>Djacobse |
||
Line 53: | Line 53: | ||
<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> | ||
<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>'''Take 10 uL as for qPCR'''</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> | ||
<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> | ||
<li>'''Take 10 uL as for qPCR'''</li> | |||
</ol> | </ol> | ||
<li>Latch and Padlock Hybridization</li> | <li>Latch and Padlock Hybridization</li> | ||
Line 61: | Line 63: | ||
<li>Resuspend pellet in 100 uL LPH buffer with 100 nM Padlock0201-68 and 100 nM Latch0001</li> | <li>Resuspend pellet in 100 uL LPH buffer with 100 nM Padlock0201-68 and 100 nM Latch0001</li> | ||
<li>Incubate for 30 min at 37C</li> | <li>Incubate for 30 min at 37C</li> | ||
<li>'''Take 10 uL as for qPCR'''</li> | |||
<li>Wash twice with 100 uL wash buffer by pelleting cells at 600xg for 3 min</li> | <li>Wash twice with 100 uL wash buffer by pelleting cells at 600xg for 3 min</li> | ||
<li>Incubate cells for 20 min at 40C in 150 uL stringent wash buffer</li> | <li>Incubate cells for 20 min at 40C in 150 uL stringent wash buffer</li> | ||
<li>'''Take 10 uL as for qPCR'''</li> | |||
<li>Resuspend with 20 uL 1x Amp Ligase buffer and transfer to a 0.2 mL tube</li></ol> | <li>Resuspend with 20 uL 1x Amp Ligase buffer and transfer to a 0.2 mL tube</li></ol> | ||
<li>Circularization</li> | <li>Circularization</li> | ||
Line 71: | Line 75: | ||
<li>Continued [[Daniel:Notebook/ComboLock/2016-10-22|tomorrow]]</li> | <li>Continued [[Daniel:Notebook/ComboLock/2016-10-22|tomorrow]]</li> | ||
</ol> | </ol> | ||
=Amplification Primers Test= | =Amplification Primers Test= |
Revision as of 21:15, 21 October 2016
Cell Test 3
Repeating the cell test but using the qPCR primers as a metric for efficiency throughout the experiment.
Protocol
Cell Trial
- C-Probe Hybridization
- Measure cell concentrations in cell counter
- Add cells to a new tube such that there are 100,000 cells present per sample
- 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
- Take 10 uL as for qPCR
- 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
- Take 10 uL as for qPCR
- Latch and Padlock Hybridization
- Pellet at 600xg for 3 min and remove supernatant
- Resuspend pellet in 100 uL LPH buffer with 100 nM Padlock0201-68 and 100 nM Latch0001
- Incubate for 30 min at 37C
- Take 10 uL as for qPCR
- 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
- Take 10 uL as for qPCR
- 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 for sample 1
- Incubate at 55C overnight (XX hours)
- Continued tomorrow
C Probes-VIM (C1v2+C2) | U87MG | |
Sample 1 | X | X |
Sample 2 | X | X |
Sample 3 | X | |
Sample 4 | X |
Amplification Primers Test
Repeating the experiment from yesterday with a few more conditions. Also, since that gel was saturated I'll be using lower concentrations.