Daniel:Notebook/ComboLock/2016-7-28: Difference between revisions
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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. | 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. | ||
==Buffers== | |||
'''KLN 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="180" height="31" | Reagent | |||
| width="65" | Stock | |||
| width="65" | uL added | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Amp Ligase Buffer | |||
| align="center" valign="bottom" | 10X | |||
| align="center" align="center" valign="bottom" | 2 | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| height="15" valign="bottom" | dNTPs | |||
| align="center" valign="bottom" | 100 uM | |||
| align="center" align="center" valign="bottom" | 2 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Hemo Klentaq | |||
| align="center" valign="bottom" | NA | |||
| align="center" align="center" valign="bottom" | 4 | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| height="15" valign="bottom" | Amp Ligase | |||
| align="center" valign="bottom" | 5 U/uL | |||
| align="center" align="center" valign="bottom" | 2 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | nfH2O | |||
| align="center" valign="bottom" | NA | |||
| align="center" align="center" valign="bottom" | 10 | |||
|} | |||
'''Phusion Buffer''' | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#D8E4BC;font-size:12pt;font-weight:bold" align="center" | |||
| width="200" height="40" | Reagent | |||
| width="65" | Stock Conc | |||
| width="72" | Final Amount | |||
| width="65" | 1x Vol (uL) | |||
| width="88" | MM Vol (4.1x) (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" | 32.8 | |||
|- style="background-color:#D9D9D9;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" | 2.46 | |||
|- 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" | 12.3 | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| height="15" 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" | 8.2 | |||
|- 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" | 8.2 | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| height="15" 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" | 13.12 | |||
|- 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" | 4.92 | |||
|- style="background-color:#D9D9D9;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" | 82 | |||
|} | |||
==Protocol== | ==Protocol== | ||
Line 93: | Line 198: | ||
<ol type="A"> | <ol type="A"> | ||
<li>Prepare 20 uL KLN mix</li> | <li>Prepare 20 uL KLN mix</li> | ||
<li>Add 2 uL KLN mix to 20 uL of reaction</li> | <li>Prepare 4.1x master mix of Phusion mix</li> | ||
<li>Add 20 uL Phusion mix to 20 uL of reaction for samples 2-5</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>Incubate at 55C overnight (XX hours) </li> | ||
<li>Continued [[Daniel:Notebook/ComboLock/2016-7- | <li>Continued [[Daniel:Notebook/ComboLock/2016-7-29|tomorrow]]</li> | ||
</ol> | </ol> | ||
[[Category:ComboLock]] [[Category:20160728]] |
Latest revision as of 20:41, 28 July 2016
Phusion Test[edit]
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.
Buffers[edit]
KLN Mix
Reagent | Stock | uL added |
Amp Ligase Buffer | 10X | 2 |
dNTPs | 100 uM | 2 |
Hemo Klentaq | NA | 4 |
Amp Ligase | 5 U/uL | 2 |
nfH2O | NA | 10 |
Phusion Buffer
Reagent | Stock Conc | Final Amount | 1x Vol (uL) | MM Vol (4.1x) (uL) |
NAD+ | 5 mM | 40 nmol | 8 | 32.8 |
dNTP | 1 mM | 600 pmol | 0.6 | 2.46 |
Betaine | 5 M | 15 umol | 3 | 12.3 |
10X AmpLigase Buffer | 10X | 1X | 2 | 8.2 |
Amp Ligase | 5 U/uL | 10 U | 2 | 8.2 |
Phusion HF DNA Polymerase | 2000 U/mL | 6.4U | 3.2 | 13.12 |
nf H2O | 1.2 | 4.92 | ||
Total | 20 | 82 |
Protocol[edit]
- 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
- Prepare 4.1x master mix of Phusion mix
- Add 20 uL Phusion mix to 20 uL of reaction for samples 2-5
- Add 2 uL KLN mix to 20 uL of reaction for sample 1
- 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 |