Daniel:Notebook/ComboLock/2017-2-14: Difference between revisions
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>Djacobse (Created page with "=Beads Binding Test (Started Thursday 2-9)= Back to Calendar ==Positive Control Test== [[Daniel:Noteboo...") |
>Djacobse |
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{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | {| 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" | |- style="background-color:#B1A0C7;font-size:12pt;font-weight:bold" align="center" | ||
| width=" | | width="65" height="45" | Master Mix | ||
| width=" | | width="119" | Primer Pair | ||
| width=" | | width="65" | Master Mix NoX | ||
| width=" | | width="65" | Forward Primer Amt (10 uM) | ||
| width=" | | width="65" | Reverse Primer Amt (10 uM) | ||
| width=" | | width="65" | uL nfH2O | ||
| width="65" | uL 2X Kapa SYBR Fast | |||
| width="65" | Total | |||
|- style="font-size:12pt" align="center" valign="bottom" | |- style="font-size:12pt" | ||
| align="center" | | height="15" valign="bottom" | MMT | ||
| align="center" | | | align="center" valign="bottom" | PCCT-F/PCCT-R | ||
| align="center" | | | align="center" align="center" | 11.2 | ||
| align="center" | | | align="center" align="center" | 11.2 | ||
| align="center" | | | align="center" align="center" | 11.2 | ||
|style="font-weight:bold" align="center" | | | align="center" align="center" | 235.2 | ||
| align="center" align="center" | 280 | |||
|style="font-weight:bold" align="center" align="center" | 537.6 | |||
|- style="background-color:#BFBFBF;font-size:12pt" align="center" valign="bottom" | |- style="background-color:#BFBFBF;font-size:12pt" | ||
| align="center" | | height="15" valign="bottom" | MMC1 | ||
| align="center" | | | align="center" valign="bottom" | primer24/primer4RC | ||
| align="center" | | | align="center" align="center" | 11.2 | ||
| align="center" | | | align="center" align="center" | 11.2 | ||
| align="center" | | | align="center" align="center" | 11.2 | ||
|style="font-weight:bold" align="center" | | | align="center" align="center" | 235.2 | ||
| align="center" align="center" | 280 | |||
|style="font-weight:bold" align="center" align="center" | 537.6 | |||
|- style="font-size:12pt" align="center" valign="bottom" | |- style="font-size:12pt" | ||
| align="center" | | height="15" valign="bottom" | MMC2 | ||
| align="center" | | | align="center" valign="bottom" | primer6/primer12RC | ||
| align="center" | | | align="center" align="center" | 11.2 | ||
| align="center" | | | align="center" align="center" | 11.2 | ||
| align="center" | | | align="center" align="center" | 11.2 | ||
|style="font-weight:bold" align="center" | | | align="center" align="center" | 235.2 | ||
| align="center" align="center" | 280 | |||
|style="font-weight:bold" align="center" align="center" | 537.6 | |||
|} | |} | ||
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<li>72C 20 sec</li> | <li>72C 20 sec</li> | ||
<li>plate read</li> | <li>plate read</li> | ||
<li>goto b | <li>goto b x45</li> | ||
<li>72C 2 min</li> | <li>72C 2 min</li> | ||
<li>16C hold</li> | <li>16C hold</li> | ||
</ol></ol></ol> | </ol></ol></ol> | ||
===Plate Layout=== | |||
[[Image:PlateLayout-20170214.png|600px]] | |||
[[Category:ComboLock]] [[Category:20170209]] | [[Category:ComboLock]] [[Category:20170209]] |
Revision as of 19:23, 14 February 2017
Beads Binding Test (Started Thursday 2-9)
Positive Control Test
Yesterday's results showed that the positive controls failed time and time again. Looking at the math again, by adding 2 uL 10 uM template to the positive control reactions, I was adding >100X the amount Kapa's protocol recommends. So this time I ran a dilution series that covers each of the positive controls in dilutions of undiluted to 1:10000.
- qPCR
- Make qPCR master mixes according to following recipes Master Mix Recipes
- Add 48 uL of appropriate master mix and 2 uL sample according to plate layout
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x45
- 72C 2 min
- 16C hold
Master Mix | Primer Pair | Master Mix NoX | Forward Primer Amt (10 uM) | Reverse Primer Amt (10 uM) | uL nfH2O | uL 2X Kapa SYBR Fast | Total |
MMT | PCCT-F/PCCT-R | 11.2 | 11.2 | 11.2 | 235.2 | 280 | 537.6 |
MMC1 | primer24/primer4RC | 11.2 | 11.2 | 11.2 | 235.2 | 280 | 537.6 |
MMC2 | primer6/primer12RC | 11.2 | 11.2 | 11.2 | 235.2 | 280 | 537.6 |