Daniel:Notebook/HiResChrPaint/2013-11-14

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Revision as of 02:33, 15 November 2013 by >B1lake (→‎TBE Gel)
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Positive Control 2[edit]

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I'm going to start my work on positive control 2 today, amplifying all 4 probe sets and eventually coupling them to a single dye (Alexa 488). Since I've found I work best only amplifying 2 sets at a time, I'll amplify V6S1 and V6S2 first, and amplify S3 and S4 starting tomorrow or Saturday.

qPCR Amplification[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
V6S1 (12.5x) 5 625 5 5 610 1250
V6S2 (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:2013-11-14-V6S1-V6S2.png

Nanodrop Results[edit]

Probe V6S1A V6S1B V6S1C V6S1D V6S2A V6S2B V6S2C V6S2D
ng/uL 81.5 81.2 80.1 79.2 90.1 84.9 101.2 88
ug in 40 uL 3.3 3.2 3.2 3.2 3.6 3.4 4.0 3.5
Total 12.9 14.6

DpnII Digestion[edit]

1. Reaction Table
Sample V6S1A V6S1B V6S1C V6S1D V6S2A V6S2B V6S2C V6S2D
ng/uL 81.5 81.2 80.1 79.2 90.1 84.9 101.2 88
ug in 35 uL 2.9 2.8 2.8 2.8 3.2 3.0 3.5 3.1
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 2 hours; 20 minute heat shock at 65C
3. Column Purification, elute with 30 uL EB
4. Measure concentrations in nanodrop

Nanodrop Results[edit]

Sample V6S1AB V6S1CD V6S2AB V6S2CD
ng/uL 128.6 125.8 147.2 150
ug in 30 uL 3.9 3.8 4.4 4.5
total 7.6 8.9

TBE Gel[edit]

1. Lanes; (5.5 uL TBE; 2 uL 6x buffer, 0.5 uL sample; load 7.2 uL for all)


Lane 1 2 3 4 5 6 7 8 9 10 11 12
Sample 10bp ladder V6S1AB V6S1CD V6S2AB V6S2CD
2. Run for 25 minutes at 220V
3. Stain with 3 uL SYBR gold for 2 minutes

Gel Image

File:2013-11-14-DpnII.jpg