Daniel:Notebook/ComboLock/2017-3-22: Difference between revisions
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>Djacobse |
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(15 intermediate revisions by the same user not shown) | |||
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<li>Hold at 20C</li></ol> | <li>Hold at 20C</li></ol> | ||
<li>Add 1 uL T4 DNA ligase</li> | <li>Add 1 uL T4 DNA ligase</li> | ||
<li>Incubate at 20C for 30 minutes</li></ol> | <li>Incubate at 20C for 30 minutes</li> | ||
<li>Heat kill enzyme by incubating at 65C for 15 minutes</li> | |||
<li>Add 20 uL nfH2O</li></ol> | |||
<li>TBE gel</li> | <li>TBE gel</li> | ||
<li>Mix uL TBE and uL 6X dye</li> | <li>Mix 64 uL TBE and 16 uL 6X dye</li> | ||
<li>Aliquot 10 uL on to parafilm per sample/ladder</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 2 uL reaction, 1 uL (10 uM) controls, and 1.5 uL ladder to appropriate aliquot</li> | ||
[[Image:GelLanes-20170322-Circ.png|400px|Gel lanes]] | |||
<li>Add 10 uL mix to gel lanes</li> | <li>Add 10 uL mix to gel lanes</li> | ||
<li>Run gel for 24 minutes at 230V</li> | <li>Run gel for 24 minutes at 230V</li> | ||
Line 87: | Line 92: | ||
<li>Rinse and image in gel doc</li> | <li>Rinse and image in gel doc</li> | ||
</ol> | </ol> | ||
[[Image:2017-03-22-CPProduction.png|500px]] | |||
==Protocol Part 2== | |||
That was unexpected. From what I'm seeing on [http://www.bioinformatics.nl/molbi/SimpleCloningLab/electrophoresis.htm the internet], this might be a thing. Circular DNA looks to appear much higher on the gel than it should. I can try and Bglii digest to be sure. | |||
<ol start="3"> | |||
<li>Bglii digest</li> | |||
<ol type="A"> | |||
<li>Use samples 1 (Experiment) and 4 (No phosphate) for Bglii digest</li> | |||
<li>Make the following reactions; '''Do not add Bglii yet'''</li> | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#8DB4E2;font-size:12pt;font-weight:bold" align="center" | |||
| width="220" height="30" | Reagent | |||
| width="105" | Single Rxn Vol | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Ligation Reaction Product | |||
| align="center" align="center" valign="bottom" | 4 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | Bglii Cutter Primer (10 uM) | |||
| align="center" align="center" valign="bottom" | 5 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | 10X Buffer 3.1 | |||
| align="center" align="center" valign="bottom" | 2 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | Bglii | |||
| align="center" align="center" valign="bottom" | 1 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | nfH2O | |||
| align="center" align="center" valign="bottom" | 8 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | Total | |||
|style="font-weight:bold" align="center" align="center" valign="bottom" | 20 | |||
|} | |||
<li>Use the following thermocycler program</li> | |||
<ol type="a"> | |||
<li>5 min 95C</li> | |||
<li>Ramp to 50C at 0.2C/s</li> | |||
<li>10 min 50C</li> | |||
<li>1 hr 37C; '''When this cycle starts add 1 uL Bglii'''</li> | |||
<li>Heat kill with 65C for 20 min</li></ol> | |||
</ol> | |||
<li>Qiaquick Column Purification (Circular product reaction from part 1)</li> | |||
<ol type="A"> | |||
<li>Add 200 uL PB to sample</li> | |||
<li>Centrifuge at 13000rpm for 1 minute</li> | |||
<li>Dump flow through and add 700 uL PE buffer</li> | |||
<li>Centrifuge at 13000rpm for 1 minute</li> | |||
<li>Dump flow through and add 700 uL PE buffer</li> | |||
<li>Centrifuge at 13000rpm for 1 minute</li> | |||
<li>Dump flow through and centrifuge empty for 2 min at 13000 rpm</li> | |||
<li>Add 50 uL nfH2O and centrifuge for 2 min at 13000 rpm</li></ol> | |||
<li>TBE Gel II</li> | |||
<ol type="A"> | |||
<li>Mix 80 uL TBE and 20 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> | |||
[[Image:GelLanes2-20170322-Circ.png|400px|Gel lanes]] | |||
<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></ol> | |||
[[Image:2017-03-22-CPProduction-2.png|500px]] | |||
[[Category:ComboLock]] [[Category:20170322]] |
Latest revision as of 22:05, 23 March 2017
Circular Product Production[edit]
The goal of this will be to produce a large amount of circular product for use in testing.
Protocol[edit]
- 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
- Heat kill enzyme by incubating at 65C for 15 minutes
- Add 20 uL nfH2O
- TBE gel
- Mix 64 uL TBE and 16 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 Gel lanes
- 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 | 4 |
PosCon Circ Latch Oligo | 100 uM | 200 pmol | 1 |
Padlock 0601 oligo | 100 uM | 200 pmol | 1 |
C4 Positive Control oligo | 100 uM | 200 pmol | 1 |
C2 positive Control oligo | 100 uM | 200 pmol | 1 |
T4 DNA Ligase | 5 U/uL | 5 U | 1 |
nfH2O | NA | NA | 11 |
Total | 20 |
File:2017-03-22-CPProduction.png
Protocol Part 2[edit]
That was unexpected. From what I'm seeing on the internet, this might be a thing. Circular DNA looks to appear much higher on the gel than it should. I can try and Bglii digest to be sure.
- Bglii digest
- Use samples 1 (Experiment) and 4 (No phosphate) for Bglii digest
- Make the following reactions; Do not add Bglii yet
- Use the following thermocycler program
- 5 min 95C
- Ramp to 50C at 0.2C/s
- 10 min 50C
- 1 hr 37C; When this cycle starts add 1 uL Bglii
- Heat kill with 65C for 20 min
- Qiaquick Column Purification (Circular product reaction from part 1)
- Add 200 uL PB to sample
- Centrifuge at 13000rpm for 1 minute
- Dump flow through and add 700 uL PE buffer
- Centrifuge at 13000rpm for 1 minute
- Dump flow through and add 700 uL PE buffer
- Centrifuge at 13000rpm for 1 minute
- Dump flow through and centrifuge empty for 2 min at 13000 rpm
- Add 50 uL nfH2O and centrifuge for 2 min at 13000 rpm
- TBE Gel II
- Mix 80 uL TBE and 20 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 Gel lanes
- 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 | Single Rxn Vol |
Ligation Reaction Product | 4 |
Bglii Cutter Primer (10 uM) | 5 |
10X Buffer 3.1 | 2 |
Bglii | 1 |
nfH2O | 8 |
Total | 20 |