Daniel:Notebook/HiResChrPaint/2013-4-23
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Probe Prep (Started 4/22/2013)[edit]
Ethanol Precipitation (Continued from 4/22/2013)[edit]
4. Centrifuge samples at 14000 rpm at 4C for 25 minutes 5. Withdraw supernatant and resuspend with 750 uL chilled 75% EtOH 6. Centrifuge at 14000 rpm at 4C for 10 minutes 7. Dry samples in hood for ~15 minutes 8. Resuspend in 50 uL 9. Measure in nanodrop and store at 4C for use later
Nanodrop Results[edit]
Sample | ng/uL | uL | ng |
V4S3 | 1636.1 | 50 | 81805 |
V4S3A | 1617.4 | 50 | 80870 |
V6S3 | 1298.1 | 50 | 64905 |
V6S3A | 1217.9 | 50 | 60895 |
Discussion
These nanodrop results suggest that each sample has on the order of 80 ug of DNA. Since I have 2 tubes of each, it would imply that I actually have 160 ug of DNA for each sample! In previous amplifications my yield has been closer to 10 ug of DNA from this amplification. This is over 10x the yield compared with previous experiments then. Next experiment will be to run the Qiaquick columns, as the original protocol calls for, but with the appropriate amount of starting material for each column. Qiaquick columns supposedly hold only 10 ug, therefore I will test with ~8 ug of original sample and measure the efficiency of the column, as well as final yield.
Example Qiaquick Columns[edit]
Taking only column per tube (4 total) with ~8 uL each. This way I can measure the yield. I have two current hypotheses: (I) Previous PCRs have had this much DNA but since we immediately moved to columns, we lost anything greater than 10 ug (high yield) (II) This result is somehow erroneous, and will be corrected for with column purification (low yield) My expected results to confirm each hypothesis are in parentheses.
Protocol[edit]
1. Add 45 uL nuclease free water to 5 uL each sample 2. Qiaquick Column Purification 3. Elute with 40 uL EB
Nanodrop Results[edit]
Sample | ng/uL | uL | ng |
V4S3 | 12.1 | 40 | 484 |
V4S3A | 11.4 | 40 | 456 |
V6S3 | 10.7 | 40 | 428 |
V6S3A | 10.6 | 40 | 424 |
Discussion[edit]
This was an unfortunate result. It would suggest hypothesis (II), that there is a lot of contaminant from the PCR and the column washes it away. Will confirm results tomorrow.