Daniel:Notebook/HiResChrPaint/2013-4-22
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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)