Daniel:Notebook/ComboLock/2016-10-21: Difference between revisions
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>Djacobse (Created page with "=Amplification Primers Test= Back to Calendar ==Gel Image== [[Image:|800px]]") |
>Djacobse |
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= | =Cell Test 3= | ||
[[Daniel:Notebook/ComboLock|Back to Calendar]] | [[Daniel:Notebook/ComboLock|Back to Calendar]] | ||
Repeating the cell test but using the qPCR primers as a metric for efficiency throughout the experiment. | |||
==Protocol== | |||
<ol> | |||
<li>C-Probe Hybridization</li> | |||
<ol type="A"> | |||
<li>Measure cell concentrations in cell counter</li> | |||
<li>Add cells to a new tube such that there are 100,000 cells present 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="105" height="45" | | |||
|style="background-color:#CCC0DA;font-weight:bold" width="125" | C Probes-VIM (C1v2+C2) | |||
|style="background-color:#CCC0DA;font-weight:bold" width="65" | U87MG | |||
|- style="font-size:12pt" | |||
| height="15" | Sample 1 | |||
| align="center" | X | |||
| align="center" | X | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| height="15" | Sample 2 | |||
| align="center" | X | |||
| align="center" | X | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Sample 3 | |||
| align="center" align="center" | | |||
| align="center" | X | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| height="15" valign="bottom" | Sample 4 | |||
| align="center" | X | |||
| align="center" align="center" | | |||
|} | |||
<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>Add 100 uL 50% hematoxylin (filtered) and pellet cells via centrifugation at 600g for 3 min</li> | |||
<li>Wash once with 200 uL wash buffer 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>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>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>'''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>Incubate cells for 20 min at 40C in 200 uL stringent wash buffer</li> | |||
<li>'''Take 10 uL as for qPCR'''</li> | |||
</ol> | |||
<li>Latch and Padlock Hybridization</li> | |||
<ol type="A"> | |||
<li>Pellet at 600xg for 3 min and remove supernatant</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>'''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>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>Circularization</li> | |||
<ol type="A"> | |||
<li>Prepare 20 uL KLN mix</li> | |||
<li>Add 2 uL KLN mix to 20 uL of reaction for sample 1</li> | |||
<li>Incubate at 55C overnight (XX hours) </li> | |||
<li>Continued [[Daniel:Notebook/ComboLock/2016-10-22|tomorrow]]</li> | |||
</ol> | |||
=Amplification Primers Test= | |||
Repeating the experiment from [[Daniel:Notebook/ComboLock/2016-10-20|yesterday]] with a few more conditions. Also, since that gel was saturated I'll be using lower concentrations. | |||
==Gel Image== | ==Gel Image== | ||
[[Image:|800px]] | [[Image:2016-10-21-AmplificationPrimers2.png|800px]] | ||
[[Category:ComboLock]] [[Category:20161021]] |
Latest revision as of 18:20, 24 October 2016
Cell Test 3[edit]
Repeating the cell test but using the qPCR primers as a metric for efficiency throughout the experiment.
Protocol[edit]
- 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[edit]
Repeating the experiment from yesterday with a few more conditions. Also, since that gel was saturated I'll be using lower concentrations.