Daniel:Notebook/HiResChrPaint/2013-8-8: Difference between revisions
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===qPCR Results=== | ===qPCR Results=== | ||
[[Image:2013-8-8-S2-ULS.png|600px]] | |||
===Nanodrop Results=== | ===Nanodrop Results=== | ||
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2. Incubate samples at 37C for | 2. Incubate samples at 37C for 16 hours; 20 minute heat shock at 65C; [[Daniel:Notebook/HiResChrPaint/2013-8-9|Continued 8/9/2013]] | ||
Latest revision as of 17:00, 9 August 2013
S2 Probe Prep (ULS)[edit]
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[edit]
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
qPCR Results[edit]
Nanodrop Results[edit]
Sample | V4S2A | V4S2B | V4S2C | V4S2D | V6S2A | V6S2B | V6S2C | V6S2D |
ng/uL | 64.7 | 62.6 | 69.1 | 79.2 | 139 | 126.8 | 127.3 | 127.2 |
ug in 40 uL | 2.6 | 2.5 | 2.8 | 3.2 | 5.6 | 5.1 | 5.1 | 5.1 |
Total ug | 11.0 | 20.8 |
DpnII Digestion[edit]
1. Prepare Samples
Sample | V4S2A | V4S2B | V4S2C | V4S2D | V6S2A | V6S2B | V6S2C | V6S2D |
ng/uL | 64.7 | 62.6 | 69.1 | 79.2 | 139 | 126.8 | 127.3 | 127.2 |
ug in 35 uL | 2.3 | 2.2 | 2.4 | 2.8 | 4.9 | 4.4 | 4.5 | 4.5 |
uL 10X buffer | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 |
uL DpnII (50U/uL) | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 |
uL nfH2O | 8 | 8 | 8 | 8 | 8 | 8 | 8 | 8 |
Total | 50 | 50 | 50 | 50 | 50 | 50 | 50 | 50 |
2. Incubate samples at 37C for 16 hours; 20 minute heat shock at 65C; Continued 8/9/2013