Daniel:Notebook/ComboLock/2017-3-16: Difference between revisions
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Rolling Circle Amplification
>Djacobse (Created page with "=Circularization Comparison (T4 Poly/Ligase)= Back to Calendar ==Protocol-Part 4== <ol start="7"> <li>Exonuclease Digestion</li> <ol type="A">...") |
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<li>Heat kill by incubating for 5 min at 95C</li> | <li>Heat kill by incubating for 5 min at 95C</li> | ||
</ol> | </ol> | ||
<li>qPCR</li> | |||
<ol type="A"> | |||
<li>Make qPCR master mixes according to following recipe</li> | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#B1A0C7;font-size:12pt;font-weight:bold" align="center" | |||
| width="200" height="39" | Reagent | |||
| width="85" | Single Rxn | |||
| width="160" | MMProd (15.2X) | |||
| width="160" | MM+ (C1, 4.2X) | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Primer Pair (fwd/rev) | |||
| align="center" valign="bottom" | NA | |||
| align="center" valign="bottom" | primer12*/AmpR-Ind21 | |||
| align="center" valign="bottom" | primer24/primer4* | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | Forward Primer (10 uM) | |||
| align="center" align="center" valign="bottom" | 1 | |||
| align="center" align="center" valign="bottom" | 15.2 | |||
| align="center" align="center" valign="bottom" | 4.2 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Reverse Primer (10 uM) | |||
| align="center" align="center" valign="bottom" | 1 | |||
| align="center" align="center" valign="bottom" | 15.2 | |||
| align="center" align="center" valign="bottom" | 4.2 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | nfH2O | |||
| align="center" align="center" valign="bottom" | 21 | |||
| align="center" align="center" valign="bottom" | 319.2 | |||
| align="center" align="center" valign="bottom" | 88.2 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | 2X Kapa SYBR Fast | |||
| align="center" align="center" valign="bottom" | 25 | |||
| align="center" align="center" valign="bottom" | 380 | |||
| align="center" align="center" valign="bottom" | 105 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | Total | |||
|style="font-weight:bold" align="center" align="center" valign="bottom" | 48 | |||
|style="font-weight:bold" align="center" align="center" valign="bottom" | 729.6 | |||
|style="font-weight:bold" align="center" align="center" valign="bottom" | 201.6 | |||
|} | |||
<li>Add 48 uL master mix to each well</li> | |||
<li>Add 2 uL sample according to plate layout</li> | |||
[[Image:|600px]] | |||
<li>qPCR Cycles</li> | |||
<ol type="a"> | |||
<li>95C 3 min</li> | |||
<li>95C 3 sec</li> | |||
<li>55C 30 sec</li> | |||
<li>72C 20 sec</li> | |||
<li>plate read</li> | |||
<li>goto b x30</li> | |||
<li>72C 2 min</li> | |||
<li>16C hold</li> | |||
</ol></ol></ol> | |||
<li>Rolling Circle Amplification</li> | <li>Rolling Circle Amplification</li> | ||
<ol type="A"> | <ol type="A"> | ||
<li>Samples will be aliquoted under following rules</li> | <li>Samples will be aliquoted under following rules</li> |
Revision as of 18:44, 16 March 2017
Circularization Comparison (T4 Poly/Ligase)
Protocol-Part 4
- Exonuclease Digestion
- Mix 15 uL Exo I (20U/uL) and 3 uL ExoIII (100U/uL)
- Add 2 uL to each sample, mix by swirling pipette tip
- Incubate for 1.5 hours at 37C
- Heat kill by incubating for 5 min at 95C
- qPCR
- Make qPCR master mixes according to following recipe
- Add 48 uL master mix to each well
- Add 2 uL sample according to plate layout [[Image:|600px]]
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x30
- 72C 2 min
- 16C hold
Reagent | Single Rxn | MMProd (15.2X) | MM+ (C1, 4.2X) |
Primer Pair (fwd/rev) | NA | primer12*/AmpR-Ind21 | primer24/primer4* |
Forward Primer (10 uM) | 1 | 15.2 | 4.2 |
Reverse Primer (10 uM) | 1 | 15.2 | 4.2 |
nfH2O | 21 | 319.2 | 88.2 |
2X Kapa SYBR Fast | 25 | 380 | 105 |
Total | 48 | 729.6 | 201.6 |
- Samples will be aliquoted under following rules