Daniel:Notebook/HiResChrPaint/2013-4-22: Difference between revisions
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Purpose: Start new probe synthesis (from scratch) to better assess methods. Getting low yields on dye coupling reaction, | Purpose: Start new probe synthesis (from scratch) to better assess methods. Getting low yields on dye coupling reaction, | ||
but I think that the reason is I have much lower yields than Noi's original run. Going to try the entire synthesis, focusing on only | but I think that the reason is I have much lower yields than Noi's original run. Going to try the entire synthesis, focusing on only | ||
2 sets, V4S3 and V6S3. This way I can focus my attention better and use a more appropriately sized trial run. | 2 sets, V4S3 and V6S3. This way I can focus my attention better and use a more appropriately sized trial run. S3 was chosen because of | ||
its high yield after previous PCRs. At the end of this run I plan on painting the cells using only this set, again as a trial before the | |||
multi-colored painting. | |||
==Expansion qPCR== | ==Expansion qPCR== | ||
1. Set up 8 reactions for each of the 2 probes (16 reactions total) | 1. Set up 8 reactions for each of the 2 probes (16 reactions total) | ||
{| 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="font-size:12pt | |- style="font-size:12pt" | ||
|style="background-color:#FFFF00" align="right" width=" | |style="background-color:#FFFF00" align="right" width="114" height="15" align="right" | | ||
|style="font-weight:bold" width="65" | |style="font-weight:bold" width="65" align="center" | per rxn | ||
|style="font-weight:bold" width="65" align="center" | V4S3 | |||
|style="font-weight:bold" width="65" | |style="font-weight:bold" width="65" align="center" | V6S3 | ||
|style="font-weight:bold" width="65 | |||
|- style="font-size:12pt" align="center" | |- style="font-size:12pt" | ||
| | | height="15" align="center" | 20 nM OligoPool | ||
| align="right" align="right" | 0.2 | |||
| align="right" | | align="right" align="right" | 1.7 | ||
| align="right" align="right" | 1.7 | |||
| align="right" | |||
|- style="font-size:12pt" align="center" | |- style="font-size:12pt" | ||
| | | height="30" align="center" | 2X Kapa SYBR qPCR MM | ||
| | | align="right" align="right" | 50 | ||
| align="right" align="right" | 425 | |||
| | | align="right" align="right" | 425 | ||
|- style="font-size:12pt" align="center" | |- style="font-size:12pt" | ||
| | | height="15" align="center" | 100 uM AP1V41U | ||
| align="right" align="right" | 0.4 | |||
| align="right" | | align="right" align="right" | 3.4 | ||
| align="right" align="right" | 0 | |||
| align="right" | |||
|- style="font-size:12pt" align="center" | |- style="font-size:12pt" | ||
| | | height="15" align="center" | 100 uM AP2V4 | ||
| align="right" align="right" | 0.4 | |||
| align="right" | | align="right" align="right" | 3.4 | ||
| align="right" align="right" | 0 | |||
| align="right" | |||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
| height="15" align="center" | 100 uM AP1V61U | |||
| align="right" | 0.4 | | align="right" align="right" | 0.4 | ||
| align="right" | | align="right" align="right" | 0 | ||
| align="right" align="right" | 3.4 | |||
| align="right" | |||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
| height="15" align="center" | 100 uM AP2V6 | |||
| align="right" | 0.4 | | align="right" align="right" | 0.4 | ||
| align="right" | | align="right" align="right" | 0 | ||
| align="right" align="right" | 3.4 | |||
| align="right" | |||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
| height="15" align="center" | 2 mM aa-dUTP | |||
| align="right" | 3 | | align="right" align="right" | 3 | ||
| align="right" | | align="right" align="right" | 25.5 | ||
| align="right" align="right" | 25.5 | |||
| align="right" | |||
|- style="font-size:12pt" align="center" | |- style="font-size:12pt" | ||
| | | height="15" align="center" | H20 | ||
| align="right" align="right" | 46 | |||
| align="right" | | align="right" align="right" | 391 | ||
| align="right" align="right" | 391 | |||
| align="right" | |||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
| height="15" align="center" | Total Volume | |||
| align="right" | 100 | | align="right" align="right" | 100 | ||
| align="right" | | align="right" align="right" | 850 | ||
| align="right" align="right" | 850 | |||
| align="right" | |||
|} | |} |
Revision as of 19:55, 22 April 2013
Probe Prep S3 4/22/2013
Purpose: Start new probe synthesis (from scratch) to better assess methods. Getting low yields on dye coupling reaction, but I think that the reason is I have much lower yields than Noi's original run. Going to try the entire synthesis, focusing on only 2 sets, V4S3 and V6S3. This way I can focus my attention better and use a more appropriately sized trial run. S3 was chosen because of its high yield after previous PCRs. At the end of this run I plan on painting the cells using only this set, again as a trial before the multi-colored painting.
Expansion qPCR
1. Set up 8 reactions for each of the 2 probes (16 reactions total)
per rxn | V4S3 | V6S3 | |
20 nM OligoPool | 0.2 | 1.7 | 1.7 |
2X Kapa SYBR qPCR MM | 50 | 425 | 425 |
100 uM AP1V41U | 0.4 | 3.4 | 0 |
100 uM AP2V4 | 0.4 | 3.4 | 0 |
100 uM AP1V61U | 0.4 | 0 | 3.4 |
100 uM AP2V6 | 0.4 | 0 | 3.4 |
2 mM aa-dUTP | 3 | 25.5 | 25.5 |
H20 | 46 | 391 | 391 |
Total Volume | 100 | 850 | 850 |