Daniel:Notebook/HiResChrPaint/2013-10-22

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BSPP Control[edit]

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  • For first production of BSPP control, see 09/03/2013

Since I'm out of control group, I'm going to make some more.

qPCR[edit]

1. Reaction Table
  10 nM OligoPool 2X Kapa SYBR qPCR MM 100 uM AP1V61U-Amino 100 uM AP2V6 H20 Total Volume
per rxn 0.4 50 0.4 0.4 48.8 100
BSPP 1 (12.5x) 5 625 5 5 610 1250
BSPP 2 (12.5x) 5 625 5 5 610 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 x17
    vii. 72C 2 min
    viii. 16C hold
4. Pooled 3 samples together each (300 uL each, 4 pools per set, 8 total)
5. Column Purification, elute with 40 uL EB
6. Measure concentrations in nanodrop

qPCR Results[edit]

File:2013-10-22-BSPPControl.png

Nanodrop Results[edit]

Probe BSPP 1A BSPP 1B BSPP 1C BSPP 1D BSPP 2A BSPP 2B BSPP 2C BSPP 2D
ng/uL 97.9 94.5 96.9 91.9 90.2 92.5 90.7 82.4
ug in 40 uL 3.9 3.8 3.9 3.7 3.6 3.7 3.6 3.3
Total       15.2       14.2

DpnII Digestion[edit]

1. Reaction Table
Sample BSPP 1A BSPP 1B BSPP 1C BSPP 1D BSPP 2A BSPP 2B BSPP 2C BSPP 2D
ng/uL 97.9 94.5 96.9 91.9 90.2 92.5 90.7 82.4
ug in 35 uL 3.4 3.3 3.4 3.2 3.2 3.2 3.2 2.9
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 tomorrow