Daniel:Notebook/HiResChrPaint/2013-6-20: Difference between revisions

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Notes: *1.7x yield (in ug) of qPCR of V4S1 using 800 uL starting material; ** 2.1x yield (in ug) of qPCR of V6S1 using 800 uL starting material

Revision as of 00:11, 21 June 2013

S1 Probe Preparation

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PCR amplification

1. Reaction Table
  20 nM OligoPool 2X Kapa SYBR qPCR MM 100 uM AP1V41U 100 uM AP2V4 100 uM AP1V61U 100 uM AP2V6 2 mM aa-dUTP H20 Total Volume
per rxn 0.2 50 0.4 0.4 0.4 0.4 2.5 45.7 100
V4S1 (12.5x) 2.5 625 5 5 0 0 31 581.5 1250
V6S1 (12.5x) 2.5 625 0 0 5 5 31 581.5 1250
2. Aliquot 100 uL into each of 12 qPCR tubes 
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. Pooled 3 samples together each (300 uL each, 4 pools per set, 8 total)
5. Ethanol Precipitation, added 900 uL 100% EtOH, 30 uL 3 M NaOAc, 1 uL glycoblue
6. Incubated 2 hours; resuspend in 50 uL nfH20
7. Column Purification, elute with 40 uL EB
8. Measure concentrations in nanodrop

qPCR Results

File:V4S1-062013.png

Nanodrop Results

Sample V4S1A V4S1B V4S1C V4S1D V6S1A V6S1B V6S1C V6S1D
ng/uL 30.7 31.1 31.5 29.1 90 87.7 91.9 89.9
ug in 40 uL 1.228 1.244 1.26 1.164 3.6 3.508 3.676 3.596
Total ug       4.896*       14.38**

Notes: *1.7x yield (in ug) of qPCR of V4S1 using 800 uL starting material; ** 2.1x yield (in ug) of qPCR of V6S1 using 800 uL starting material