Daniel:Notebook/ComboLock/2016-11-7: Difference between revisions
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This experiment is designed to see if I can get mRNA targeting C-Probes to work in cells, this time with RCA. I'll be trying both VIM and SOD1, just to have two separate genes. | This experiment is designed to see if I can get mRNA targeting C-Probes to work in cells, this time with RCA. I'll be trying both VIM and SOD1, just to have two separate genes. | ||
==Buffer Prep== | |||
Need more C Probe Hybridization Buffer. I'll be freezing it down in aliquots of 500 uL. | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="font-size:12pt" valign="bottom" | |||
| align="center" width="172" height="15" | | |||
| align="center" width="65" | | |||
| align="center" width="65" | | |||
| align="center" width="65" | | |||
| align="center" width="65" | | |||
|- style="background-color:#6AFF6F;font-size:12pt;font-weight:bold" align="center" | |||
| height="30" | Reagent | |||
| Stock | |||
| Final | |||
| Dilution | |||
| Amount in 10 mL (uL) | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | SSC Buffer | |||
| align="center" valign="bottom" | 20X | |||
| align="center" valign="bottom" | 1X | |||
| align="center" align="center" valign="bottom" | 20 | |||
| align="center" align="center" valign="bottom" | 500 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | Polyvinylsulonic Acid | |||
| align="center" align="center" valign="bottom" | 100% | |||
| align="center" align="center" valign="bottom" | 2.50% | |||
| align="center" align="center" valign="bottom" | 40 | |||
| align="center" align="center" valign="bottom" | 250 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Ribonucleoside Vanadyl Complex | |||
| align="center" valign="bottom" | 200 mM | |||
| align="center" valign="bottom" | 20 mM | |||
| align="center" align="center" valign="bottom" | 10 | |||
| align="center" align="center" valign="bottom" | 1000 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | Rnasin | |||
| align="center" valign="bottom" | 40000 U/mL | |||
| align="center" valign="bottom" | 40 U/mL | |||
| align="center" align="center" valign="bottom" | 1000 | |||
| align="center" align="center" valign="bottom" | 10 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | 0.1% Tween-20 | |||
| align="center" align="center" valign="bottom" | 10% | |||
| align="center" align="center" valign="bottom" | 0.10% | |||
| align="center" align="center" valign="bottom" | 100 | |||
| align="center" align="center" valign="bottom" | 100 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | Salmon Sperm DNA | |||
| align="center" valign="bottom" | 10 mg/mL | |||
| align="center" valign="bottom" | 100 ug/mL | |||
| align="center" align="center" valign="bottom" | 100 | |||
| align="center" align="center" valign="bottom" | 100 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | DEPC Treated Water | |||
| align="center" valign="bottom" | NA | |||
| align="center" valign="bottom" | NA | |||
| align="center" valign="bottom" | NA | |||
| align="center" align="center" valign="bottom" | 8040 | |||
|} | |||
==Protocol== | ==Protocol== | ||
'''Sample Matrix''' | |||
{| 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="92" height="45" | | |||
|style="background-color:#CCC0DA;font-weight:bold" width="65" | C Probes-VIM /SOD1 (C1v2+C2) | |||
|style="background-color:#CCC0DA;font-weight:bold" width="65" | U87MG | |||
|- style="font-size:12pt" | |||
| height="15" | Sample 1 (AB) | |||
| align="center" | X | |||
| align="center" | X | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| height="15" | Sample 2 (AB) | |||
| align="center" | X | |||
| align="center" | X | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Sample 3 (AB) | |||
| align="center" align="center" | | |||
| align="center" | X | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| height="15" valign="bottom" | Sample 4 (AB) | |||
| align="center" | X | |||
| align="center" align="center" | | |||
|} | |||
<ol> | <ol> | ||
Line 33: | Line 134: | ||
<li>Circularization</li> | <li>Circularization</li> | ||
<ol type="A"> | <ol type="A"> | ||
<li>Prepare | <li>Prepare 8.2X uL Phusion mix</li> | ||
<li>Add | <li>Add 20 uL Phusion mix to 20 uL of reaction for sample 1</li> | ||
<li>Incubate at 55C | <li>Incubate at 55C for 2 hours</li> | ||
<li>Continued [[Daniel:Notebook/ComboLock/2016-10-22|tomorrow]]</li> | <li>Continued [[Daniel:Notebook/ComboLock/2016-10-22|tomorrow]]</li> | ||
</ol> | </ol> | ||
===Buffers=== | |||
'''Phusion Mix''' | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#C4D79B;font-size:12pt;font-weight:bold" align="center" | |||
| width="107" height="30" | Reagent | |||
| width="65" | Stock Conc | |||
| width="65" | Final Amount | |||
| width="65" | 1x Vol (uL) | |||
| width="65" | MM Vol (8.2x) (uL) | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | NAD+ | |||
| align="center" valign="bottom" | 5 mM | |||
| align="center" valign="bottom" | 40 nmol | |||
| align="center" align="center" valign="bottom" | 8 | |||
| align="center" align="center" valign="bottom" | 65.6 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | dNTP | |||
| align="center" valign="bottom" | 1 mM | |||
| align="center" valign="bottom" | 600 pmol | |||
| align="center" align="center" valign="bottom" | 0.6 | |||
| align="center" align="center" valign="bottom" | 4.92 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Betaine | |||
| align="center" valign="bottom" | 5 M | |||
| align="center" valign="bottom" | 15 umol | |||
| align="center" align="center" valign="bottom" | 3 | |||
| align="center" align="center" valign="bottom" | 24.6 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="30" valign="bottom" | 10X AmpLigase Buffer | |||
| align="center" valign="bottom" | 10X | |||
| align="center" valign="bottom" | 1X | |||
| align="center" align="center" valign="bottom" | 2 | |||
| align="center" align="center" valign="bottom" | 16.4 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Amp Ligase | |||
| align="center" valign="bottom" | 5 U/uL | |||
| align="center" valign="bottom" | 10 U | |||
| align="center" align="center" valign="bottom" | 2 | |||
| align="center" align="center" valign="bottom" | 16.4 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="30" valign="bottom" | Phusion HF DNA Polymerase | |||
| align="center" valign="bottom" | 2000 U/mL | |||
| align="center" valign="bottom" | 6.4U | |||
| align="center" align="center" valign="bottom" | 3.2 | |||
| align="center" align="center" valign="bottom" | 26.24 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | nf H2O | |||
| align="center" align="center" valign="bottom" | | |||
| align="center" align="center" valign="bottom" | | |||
| align="center" align="center" valign="bottom" | 1.2 | |||
| align="center" align="center" valign="bottom" | 9.84 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | Total | |||
| align="center" align="center" valign="bottom" | | |||
| align="center" align="center" valign="bottom" | | |||
| align="center" align="center" valign="bottom" | 20 | |||
| align="center" align="center" valign="bottom" | 164 | |||
|} |
Revision as of 18:03, 7 November 2016
Cell Test with RCA
This experiment is designed to see if I can get mRNA targeting C-Probes to work in cells, this time with RCA. I'll be trying both VIM and SOD1, just to have two separate genes.
Buffer Prep
Need more C Probe Hybridization Buffer. I'll be freezing it down in aliquots of 500 uL.
Reagent | Stock | Final | Dilution | Amount in 10 mL (uL) |
SSC Buffer | 20X | 1X | 20 | 500 |
Polyvinylsulonic Acid | 100% | 2.50% | 40 | 250 |
Ribonucleoside Vanadyl Complex | 200 mM | 20 mM | 10 | 1000 |
Rnasin | 40000 U/mL | 40 U/mL | 1000 | 10 |
0.1% Tween-20 | 10% | 0.10% | 100 | 100 |
Salmon Sperm DNA | 10 mg/mL | 100 ug/mL | 100 | 100 |
DEPC Treated Water | NA | NA | NA | 8040 |
Protocol
Sample Matrix
C Probes-VIM /SOD1 (C1v2+C2) | U87MG | |
Sample 1 (AB) | X | X |
Sample 2 (AB) | X | X |
Sample 3 (AB) | X | |
Sample 4 (AB) | X |
- Cell Preparation
- 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 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 8.2X uL Phusion mix
- Add 20 uL Phusion mix to 20 uL of reaction for sample 1
- Incubate at 55C for 2 hours
- Continued tomorrow
Buffers
Phusion Mix
Reagent | Stock Conc | Final Amount | 1x Vol (uL) | MM Vol (8.2x) (uL) |
NAD+ | 5 mM | 40 nmol | 8 | 65.6 |
dNTP | 1 mM | 600 pmol | 0.6 | 4.92 |
Betaine | 5 M | 15 umol | 3 | 24.6 |
10X AmpLigase Buffer | 10X | 1X | 2 | 16.4 |
Amp Ligase | 5 U/uL | 10 U | 2 | 16.4 |
Phusion HF DNA Polymerase | 2000 U/mL | 6.4U | 3.2 | 26.24 |
nf H2O | 1.2 | 9.84 | ||
Total | 20 | 164 |