Daniel:Notebook/HiResChrPaint/2013-11-14
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Positive Control 2[edit]
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]
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