Daniel:Notebook/ComboLock/2016-10-18: Difference between revisions
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===Results=== | ===Results=== | ||
[[Category:ComboLock]] [[Category:20161017]] | <gallery perrow=2 heights=300px widths=300px> | ||
File:20161018-qPCR-CellTestPadlock0201.png|qPCR Curve | |||
File:2016-10-18-CellTest-Padlock0201.png|Gel Image | |||
</gallery> | |||
=Cell Trial 2= | |||
There may have been issues with the previous run, so I'm going to repeat as is. | |||
<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 100 uL 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>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> | |||
</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>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>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/2016-10-19|tomorrow]]</li> | |||
</ol> | |||
[[Category:ComboLock]] [[Category:20161017]] [[Category:20161018]] |
Latest revision as of 18:17, 24 October 2016
Cell Trial Padlock0201 (Started Yesterday)[edit]
Protocol[edit]
- Circularization
- Heat inactivate enzyme by incubating for 2 minutes at 94C
- Exonuclease Digestion
- Prepare exonuclease I/III mix by mixing 7.5 uL exonuclease I (20 units/μL) and 1.5 uL exonuclease III (100 units/μL) in 1:1 ratio
- Add 2 μL of exonuclease I/III mix to reaction
- Mix the reaction by swirling pipette around the well 5 times
- Incubate reaction at 37 ºC for 2 hours
- Heat inactivate enzyme by incubating at 94C for 5 minutes
- qPCR
- Prepare the following master mix
- 89.25 uL nfH2O
- 2.55 uL 100 uM AmpF6.4Sol
- 127.5 uL Kapa SYBR Fast MM
- Add 43 uL master mix to each well
- Add sample and AmpR indexes according to sample table below
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x24
- 72C 2 min
- 16C hold
- TBE Gel
- Mix 56 uL TBE, 14 uL 6x loading dye
- Aliquot 10 uL per sample/ladder lane onto parafilm
- Add 2 uL of sample or ladder to correct drop
- Load 10 uL in to well
- Run gel for 22 minutes at 250V
- Open gel and stain with 3 uL SYBR Gold for 3 minutes
- Rinse gel and image in gel doc
Sample | Condition | Lane | AmpR Index | Sample Vol (uL) | 2X Kapa SYBR qPCR MM | 100 uM AmpF6.4Sol | 10 uM AmpR6.3-IndXX | H2O | Total Volume (uL) |
Sample 1 | Exp. Rep 1 | A1 | 20 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 2 | Exp. Rep 2 | B1 | 20 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 3 | No C Probes | C1 | 21 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 4 | No Cells | D1 | 22 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
NTC | No qPCR sample | E1 | 23 | 0 | 25 | 0.5 | 5 | 19.5 | 50 |
Results[edit]
- 20161018-qPCR-CellTestPadlock0201.png
qPCR Curve
- 2016-10-18-CellTest-Padlock0201.png
Gel Image
Cell Trial 2[edit]
There may have been issues with the previous run, so I'm going to repeat as is.
- 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 100 uL 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 Padlock0201-68 and 100 nM Latch0001
- 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 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 |