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" | per rxn | |style="font-weight:bold" width="65" align="center" | per rxn | ||
|style="font-weight:bold" width="65" | |style="font-weight:bold" width="65" align="center" | V4S3 | ||
|style="font-weight:bold" width="65" align="center" | V6S3 | |||
|style="font-weight:bold" width="65" | |||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
| height="15" align="center" | 20 nM OligoPool | |||
| align="right" | 0.2 | | 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" | |- style="font-size:12pt" | ||
| height="30" align="center" | 2X Kapa SYBR qPCR MM | |||
| align="right" | 50 | | align="right" align="right" | 50 | ||
| align="right" | | align="right" align="right" | 425 | ||
| align="right" align="right" | 425 | |||
| align="right" | |||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
| height="15" align="center" | 100 uM AP1V41U | |||
| align="right" | 0.4 | | 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 AP2V4 | |||
| align="right" | 0.4 | | 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 AP1V61U | ||
| 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" align="center" | |- style="font-size:12pt" | ||
| | | height="15" align="center" | 100 uM AP2V6 | ||
| 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" align="center" | |- style="font-size:12pt" | ||
| | | height="15" align="center" | 50 mM aa-dUTP | ||
| align="right" align="right" | 1/8 | |||
| align="right" | | align="right" align="right" | 1 | ||
| align="right" align="right" | 1 | |||
| 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" | 415 | |||
| | | align="right" align="right" | 415 | ||
|- style="font-size:12pt" align="center" | |- style="font-size:12pt" | ||
| | | height="15" align="center" | Total Volume | ||
| align="right" align="right" | 100 | |||
| align="right" | | align="right" align="right" | 850 | ||
| align="right" align="right" | 850 | |||
| align="right" | |||
| | |} | ||
2. qPCR | |||
95 C 30 seconds | |||
Repeat 16x | |||
95 C 3 seconds | |||
55 C 30 seconds | |||
72 C 20 seconds | |||
Read Plate | |||
72 C 2 min | |||
15 C Hold | |||
==Ethanol Precipitation== | |||
1. Pool half samples for given probe set into 1.5 mL eppendorf tube (4 tubes, 400 uL each) | |||
2. To each 1.5 mL eppendorf add: | |||
1000 uL EtOH (Rec 2.5X) | |||
40 uL 3M NaOAc (Rec 0.1X) | |||
5 uL Glycoblue (Rec 1/300X) | |||
3. Freeze tubes overnight at -80C ([[Daniel:Notebook/HiResChrPaint/2013-4-23|Continued on 4/23/2103]]) | |||
| |
Latest revision as of 22:51, 22 April 2013
Probe Prep S3 4/22/2013[edit]
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[edit]
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 |
50 mM aa-dUTP | 1/8 | 1 | 1 |
H20 | 46 | 415 | 415 |
Total Volume | 100 | 850 | 850 |
2. qPCR 95 C 30 seconds Repeat 16x 95 C 3 seconds 55 C 30 seconds 72 C 20 seconds Read Plate 72 C 2 min 15 C Hold
Ethanol Precipitation[edit]
1. Pool half samples for given probe set into 1.5 mL eppendorf tube (4 tubes, 400 uL each) 2. To each 1.5 mL eppendorf add: 1000 uL EtOH (Rec 2.5X) 40 uL 3M NaOAc (Rec 0.1X) 5 uL Glycoblue (Rec 1/300X) 3. Freeze tubes overnight at -80C (Continued on 4/23/2103)