Daniel:Notebook/HiResChrPaint/2013-11-15
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Positive Control (Started 11/14/2013)[edit]
Lambda Exonuclease Digestion[edit]
1. Reaction table
Sample | V6S1AB | V6S1CD | V6S2AB | V6S2CD |
ng/uL | 128.6 | 125.8 | 147.2 | 150 |
ug in 25 uL | 3.2 | 3.1 | 3.7 | 3.8 |
uL added | 25 | 25 | 25 | 25 |
uL 10x buffer | 5 | 5 | 5 | 5 |
uL Lambda Exonuclease (5U/uL) | 3 | 3 | 3 | 3 |
uL nfH2O | 17 | 17 | 17 | 17 |
total | 50 | 50 | 50 | 50 |
2. Incubate for 1 hour at 37C followed by 10 minute heat inactivation at 75C 3. Pool samples 4. ssDNA column purification, elute with 20 uL nfH2O 5. Measure Concentration in Nanodrop
ssNanodrop Results[edit]
Sample | ng/uL | ug in 20 uL |
V6S1 | 103.7 | 2.1 |
V6S2 | 226.9 | 4.5 |
Positive Control 2 (V6S3 & V6S4)[edit]
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 |
V6S3 (12.5x) | 5 | 625 | 5 | 5 | 610 | 1250 |
V6S4 (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 | V6S3A | V6S3B | V6S3C | V6S3D | V6S4A | V6S4B | V6S4C | V6S4D |
ng/uL | 108.7 | 107.4 | 108.1 | 103.8 | 113.7 | 111.4 | 110.4 | 113.3 |
ug in 40 uL | 4.3 | 4.3 | 4.3 | 4.2 | 4.5 | 4.5 | 4.4 | 4.5 |
Total | 17.1 | 18.0 |
DpnII Digestion[edit]
1. Reaction Table
Sample | V6S3A | V6S3B | V6S3C | V6S3D | V6S4A | V6S4B | V6S4C | V6S4D |
ng/uL | 108.7 | 107.4 | 108.1 | 103.8 | 113.7 | 111.4 | 110.4 | 113.3 |
ug in 35 uL | 3.8 | 3.8 | 3.8 | 3.6 | 4.0 | 3.9 | 3.9 | 4.0 |
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 | V6S3AB | V6S3CD | V6S4AB | V6S4CD |
ng/uL | 169.9 | 180 | 169.4 | 180.1 |
ug in 30 uL | 5.1 | 5.4 | 5.1 | 5.4 |
total | 10.5 | 10.5 |
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 | V6S3AB | V6S3CD | V6S4AB | V6S4CD |
2. Run for 25 minutes at 220V 3. Stain with 3 uL SYBR gold for 2 minutes
Gel Image