Daniel:Notebook/HiResChrPaint/2013-8-8: Difference between revisions
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>Djacobse (Created page with "=S2 Probe Prep (ULS)= Back to Calendar I have some more Alexa 488 from the ULYSIS kits left, and they recommend using it within a month. S...") |
>Djacobse |
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[[Daniel:Notebook/HiResChrPaint|Back to Calendar]] | [[Daniel:Notebook/HiResChrPaint|Back to Calendar]] | ||
I have some more Alexa 488 from the ULYSIS kits left, and they recommend using it within a month. Since it has been almost a month since I opened the bottle, I guess I should make more ( | I have some more Alexa 488 from the ULYSIS kits left, and they recommend using it within a month. Since it has been almost a month since I opened the bottle, I guess I should make more (once coupled the dye is stable for several more months). Besides, I could use the ULS labeled probe as a comparison for future FISH experiments. | ||
==qPCR Amplification== | ==qPCR Amplification== |
Revision as of 21:27, 8 August 2013
S2 Probe Prep (ULS)
I have some more Alexa 488 from the ULYSIS kits left, and they recommend using it within a month. Since it has been almost a month since I opened the bottle, I guess I should make more (once coupled the dye is stable for several more months). Besides, I could use the ULS labeled probe as a comparison for future FISH experiments.
qPCR Amplification
1. Set up master reactions according to table
20 nM OligoPool | 2X Kapa SYBR qPCR MM | 100 uM AP1V41U | 100 uM AP2V4 | 100 uM AP1V61U | 100 uM AP2V6 | 50 mM aa-dUTP | H20 | Total Volume | |
per rxn | 0.2 | 50 | 0.4 | 0.4 | 0.4 | 0.4 | 0 | 48.2 | 100 |
V4S2 (12.5x) | 2.5 | 625 | 5 | 5 | 0 | 0 | 0 | 612.5 | 1250 |
V6S2 (12.5x) | 2.5 | 625 | 0 | 0 | 5 | 5 | 0 | 612.5 | 1250 |
2. Aliquot 100 uL into each of 12 qPCR tubes (24 in total for both reactions) 3. PCR protocol: i. PCR 3 min 95C ii. 95C 3 sec iii. 55C 30 sec iv. 72C 20 sec v. plate read vi. goto ii vii. 72C 2 min viii. 16C hold 4. Pool 3 samples together each (300 uL each, 4 pools per set, 8 total) 5. Column puficiation, elute with 40 uL EB 6. Measure in Nanodrop