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===

Revision as of 16:28, 9 August 2013

S2 Probe Prep (ULS)

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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

qPCR Results

File:2013-8-8-S2-ULS.png

Nanodrop Results

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

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
3. Column purification, elute with 30 uL EB
4. Measure in Nanodrop