Daniel:Notebook/HiResChrPaint/2013-7-17
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S3 Probe Preparation (Started 7/16/2013)[edit]
DpnII Digestion[edit]
3. Column Purification, elute with 30 uL EB 4. Measure concentrations in nanodrop
Nanodrop Results[edit]
Sample | V4S3A | V4S3B | V4S3C | V4S3D | V6S3A | V6S3B | V6S3C | V6S3D |
ng/uL | 84.9 | 82.3 | 85.4 | 86.4 | 107.6 | 98.2 | 107.4 | 109.2 |
ug in 30 uL | 2.5 | 2.5 | 2.6 | 2.6 | 3.2 | 2.9 | 3.2 | 3.3 |
total ug | 10.2 | 12.7 |
DpnII Digest gel[edit]
1. Lanes; (5.5 uL TBE; 2 uL 6x buffer, 0.5 uL sample; load 7 uL for all)
Lane | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
Sample | 10bp ladder | V4S3A | V4S3B | V4S3C | V4S3D | V6S3A | V6S3B | V6S3C | V6S3D |
2. Run for 25 minutes at 250V
Gel Image
Lambda Exonuclease Digestion[edit]
1. Reaction table
Sample | V4S3A | V4S3B | V4S3C | V4S3D | V6S3A | V6S3B | V6S3C | V6S3D |
ng/uL | 84.9 | 82.3 | 85.4 | 86.4 | 107.6 | 98.2 | 107.4 | 109.2 |
ug in 25 uL | 2.1 | 2.1 | 2.1 | 2.2 | 2.7 | 2.5 | 2.7 | 2.7 |
uL added | 25 | 25 | 25 | 25 | 25 | 25 | 25 | 25 |
uL 10x buffer | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 |
uL Lambda Exonuclease (5U/uL) | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 |
uL nfH2O | 17 | 17 | 17 | 17 | 17 | 17 | 17 | 17 |
total | 50 | 50 | 50 | 50 | 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 5. Measure Concentration in Nanodrop (different from before) 6. Dry samples in vaccuum centrifuge
Nanodrop Results[edit]
Sample | V4S3 | V6S3 |
ng/uL | 166 | 228.1 |
ug in 20 uL | 3.3 | 4.6 |
Dye Coupling[edit]
- Following ARES Protocol
For each
1. Warm DMSO and bicarbonate labeling buffer and nuclease free water to room temperature 2. Resuspend DNA in 5 uL nfH20 3. Add 5 uL DNA and 3 uL of sodium bicarbonate buffer in PCR tube 4. Denature samples for 5 minutes at 95C, then snap cool using ice box 5. Dissolve 1 vial of reactive dye in 2 uL solvent; vortex ≥10 seconds *dye cannot be saved for later use. Use immediately! 6. Add 2 uL dye to sample 7. Incubate in the dark for 1 hour 8. Add 10 uL nfH20 to sample 9. Purify using Centri Sep column 10. Measure concentrations using Microarray setting in nanodrop
Labeling Results[edit]
ng/uL DNA | pmol/uL dye | ug DNA in 15 uL | pmol/uL DNA | dye/probe | dye/100 bp | Base:Dye Ratio (:1) | |
V4S3 | 140.3 | 7.5 | 2.1 | 5.2 | 1.4 | 1.8 | 57 |
V6S3 | 142.9 | 7.5 | 2.1 | 5.3 | 1.4 | 1.7 | 58 |
Discussion on S3 Probe Preparation[edit]
This probe prep took me two days. By the end I have approximately 2 ug of DNA per sample, with labeling ratios of around 50:1 bp:dye.