Daniel:Notebook/ComboLock/2017-3-22: Difference between revisions
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<ol> | <ol> | ||
<li>Ligation</li> | <li>Ligation</li> | ||
<ol type="A"> | |||
<li>Make reaction mixture according to table; '''Do not add ligase yet'''</li> | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#66FFFF;font-size:12pt;font-weight:bold" align="center" | |||
| width="147" height="36" | Reagent | |||
| width="65" | Stock Conc | |||
| width="98" | Final Conc./Amount | |||
| width="106" | uL added | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | T4 Ligase Buffer | |||
| align="center" | 5X | |||
| align="center" | 1X | |||
| align="center" align="center" | 10 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | PosCon Circ Latch Oligo | |||
| align="center" | 100 uM | |||
| align="center" | 500 umol | |||
| align="center" align="center" | 5 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Padlock 0601 oligo | |||
| align="center" | 100 uM | |||
| align="center" | 500 umol | |||
| align="center" align="center" | 5 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | C4 Positive Control oligo | |||
| align="center" | 100 uM | |||
| align="center" | 500 umol | |||
| align="center" align="center" | 5 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | C2 positive Control oligo | |||
| align="center" | 100 uM | |||
| align="center" | 500 umol | |||
| align="center" align="center" | 5 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | T4 DNA Ligase | |||
| align="center" | 5 U/uL | |||
| align="center" | 5 U | |||
| align="center" align="center" | 1 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | nfH2O | |||
| align="center" | NA | |||
| align="center" | NA | |||
| align="center" align="center" | 19 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | Total | |||
| align="center" align="center" | | |||
| align="center" align="center" | | |||
|style="font-weight:bold" align="center" align="center" | 50 | |||
|} | |||
<li>Use following thermocycler program</li> | |||
<ol type="a"> | |||
<li>Heat mixture to 95C for 5 min</li> | |||
<li>Lower temp by 0.2C per second to 55C</li> | |||
<li>Hold mixture at 55C for 15 minutes</li> | |||
<li>Lower temp to 20C by reducing 0.2C per second</li> | |||
<li>Hold at 20C</li></ol> | |||
<li>Add 1 uL T4 DNA ligase</li> | |||
<li>Incubate at 20C for 30 minutes</li></ol> | |||
<li>TBE gel</li> | |||
<li>Mix uL TBE and uL 6X dye</li> | |||
<li>Aliquot 10 uL on to parafilm per sample/ladder</li> | |||
<li>Add 2 uL reaction, 1 uL (10 uM) controls, and 1.5 uL ladder to appropriate aliquot</li> | |||
<li>Add 10 uL mix to gel lanes</li> | |||
<li>Run gel for 24 minutes at 230V</li> | |||
<li>Stain with 2 uL SYBR gold for 3 minutes</li> | |||
<li>Rinse and image in gel doc</li> | |||
</ol> |
Revision as of 18:06, 22 March 2017
Circular Product Production
The goal of this will be to produce a large amount of circular product for use in testing.
Protocol
- Ligation
- Make reaction mixture according to table; Do not add ligase yet
- Use following thermocycler program
- Heat mixture to 95C for 5 min
- Lower temp by 0.2C per second to 55C
- Hold mixture at 55C for 15 minutes
- Lower temp to 20C by reducing 0.2C per second
- Hold at 20C
- Add 1 uL T4 DNA ligase
- Incubate at 20C for 30 minutes
- TBE gel
- Mix uL TBE and uL 6X dye
- Aliquot 10 uL on to parafilm per sample/ladder
- Add 2 uL reaction, 1 uL (10 uM) controls, and 1.5 uL ladder to appropriate aliquot
- Add 10 uL mix to gel lanes
- Run gel for 24 minutes at 230V
- Stain with 2 uL SYBR gold for 3 minutes
- Rinse and image in gel doc
Reagent | Stock Conc | Final Conc./Amount | uL added |
T4 Ligase Buffer | 5X | 1X | 10 |
PosCon Circ Latch Oligo | 100 uM | 500 umol | 5 |
Padlock 0601 oligo | 100 uM | 500 umol | 5 |
C4 Positive Control oligo | 100 uM | 500 umol | 5 |
C2 positive Control oligo | 100 uM | 500 umol | 5 |
T4 DNA Ligase | 5 U/uL | 5 U | 1 |
nfH2O | NA | NA | 19 |
Total | 50 |