Daniel:Notebook/ComboLock/2017-1-25: Difference between revisions
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>Djacobse (Created page with "==Protocol== <li>qPCR</li> <ol type="A"> <li>Make qPCR master mix according to following recipe</li> <ol type="a"> <li>361.2 uL nfH2O</li> <li>17.2 uL AmpF-CLv3</li> <li>17.2...") |
>Djacobse |
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=Circularization Test (Started [[Daniel:Notebook/ComboLock/2017-1-23|Monday]])= | |||
[[Daniel:Notebook/ComboLock|Back to Calendar]] | |||
==Protocol== | ==Protocol== | ||
<ol> | |||
<li>qPCR</li> | <li>qPCR</li> | ||
<ol type="A"> | <ol type="A"> | ||
Line 11: | Line 16: | ||
<li>Add 48 uL master mix to each well</li> | <li>Add 48 uL master mix to each well</li> | ||
<li>Add 2 uL sample according to table</li> | <li>Add 2 uL sample according to table</li> | ||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#92CDDC;font-size:12pt;font-weight:bold" align="center" | |||
| width="100" height="45" | Sample | |||
| width="85" | Product Amt | |||
| width="85" | Pre/Post RCA | |||
| width="65" | Lane | |||
| width="65" | Sample Vol (uL) | |||
| width="65" | 2X Kapa SYBR qPCR MM | |||
| width="65" | 10 uM Forward Primer | |||
| width="65" | 10 uM Reverse Primer | |||
| width="65" | H2O | |||
| width="65" | Total Volume (uL) | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 0A | |||
| align="center" | 1pmol | |||
| align="center" | Pre | |||
| align="center" | A1 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 0B | |||
| align="center" | 1pmol | |||
| align="center" | Pre | |||
| align="center" | A2 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 2A | |||
| align="center" | 10 fmol | |||
| align="center" | Pre | |||
| align="center" | A3 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | Sample 2B | |||
| align="center" valign="bottom" | 10 fmol | |||
| align="center" | Pre | |||
| align="center" | A4 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 4A | |||
| align="center" | 100 amol | |||
| align="center" | Pre | |||
| align="center" | A5 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 4B | |||
| align="center" | 100 amol | |||
| align="center" | Pre | |||
| align="center" | A6 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 6A | |||
| align="center" | 1 amol | |||
| align="center" | Pre | |||
| align="center" | A7 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 6B | |||
| align="center" | 1 amol | |||
| align="center" | Pre | |||
| align="center" | A8 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 0AX | |||
| align="center" | 1pmol | |||
| align="center" | Post | |||
| align="center" | H1 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="background-color:#DA9694;font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 0BX | |||
| align="center" | 1pmol | |||
| align="center" | Post | |||
| align="center" | H2 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 2AX | |||
| align="center" | 10 fmol | |||
| align="center" | Post | |||
| align="center" | H3 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="background-color:#DA9694;font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | Sample 2BX | |||
| align="center" valign="bottom" | 10 fmol | |||
| align="center" | Post | |||
| align="center" | H4 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 4AX | |||
| align="center" | 100 amol | |||
| align="center" | Post | |||
| align="center" | H5 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="background-color:#DA9694;font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 4BX | |||
| align="center" | 100 amol | |||
| align="center" | Post | |||
| align="center" | H6 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 6AX | |||
| align="center" | 1 amol | |||
| align="center" | Post | |||
| align="center" | H7 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="background-color:#DA9694;font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 6BX | |||
| align="center" | 1 amol | |||
| align="center" | Post | |||
| align="center" | H8 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|} | |||
<li>qPCR Cycles</li> | <li>qPCR Cycles</li> | ||
<ol type="a"> | <ol type="a"> |
Revision as of 16:42, 25 January 2017
Circularization Test (Started Monday)
Protocol
- qPCR
- Make qPCR master mix according to following recipe
- 361.2 uL nfH2O
- 17.2 uL AmpF-CLv3
- 17.2 uL AmpR-CLv3
- 430 uL Kapa SYBR Fast
- Add 48 uL master mix to each well
- Add 2 uL sample according to table
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x24
- 72C 2 min
- 16C hold
- TBE Gel
- Mix 80 uL TBE, 20 uL 6x loading dye
- Aliquot 10 uL per sample/ladder lane onto parafilm
- Add 2 uL of sample or ladder to correct drop
- Load 10 uL in to well
- Run gel for 22 minutes at 250V
- Open gel and stain with 3 uL SYBR Gold for 3 minutes
- Rinse gel and image in gel doc
Sample | Product Amt | Pre/Post RCA | Lane | Sample Vol (uL) | 2X Kapa SYBR qPCR MM | 10 uM Forward Primer | 10 uM Reverse Primer | H2O | Total Volume (uL) |
Sample 0A | 1pmol | Pre | A1 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 0B | 1pmol | Pre | A2 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2A | 10 fmol | Pre | A3 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2B | 10 fmol | Pre | A4 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 4A | 100 amol | Pre | A5 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 4B | 100 amol | Pre | A6 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 6A | 1 amol | Pre | A7 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 6B | 1 amol | Pre | A8 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 0AX | 1pmol | Post | H1 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 0BX | 1pmol | Post | H2 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2AX | 10 fmol | Post | H3 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2BX | 10 fmol | Post | H4 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 4AX | 100 amol | Post | H5 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 4BX | 100 amol | Post | H6 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 6AX | 1 amol | Post | H7 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 6BX | 1 amol | Post | H8 | 2 | 25 | 1 | 1 | 21 | 50 |