Daniel:Notebook/HiResChrPaint/2014-7-9
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Strand Control (Started 07/08/2014)[edit]
DpnII Digest (from 07/08/2014)[edit]
- Column Purification, elute with 40 uL
- Measure concentration in nanodrop
Nanodrop Results[edit]
Sample | V6S2AB | V6S2CD | V6S3AB | V6S3CD |
ng/uL | 122.4 | 110.7 | 137 | 133.5 |
ug in 40 uL | 4.9 | 4.4 | 5.5 | 5.3 |
ug total | 9.3 | 10.8 | ||
Yield (Max ~80%) | 71.18 | 71.18 |
Scary that both of the columns yielded the same percent (I double checked the math). As a note, taking into account that the theoretical maximum yield is 80% (since we remove 20bp out of 100 on each strand), this is approximately a 90% yield from the column.
TBE Gel[edit]
Gel Image
Lambda Exonuclease Digestion[edit]
- Reaction Table
Sample | V6S2AB | V6S2CD | V6S3AB | V6S3CD |
ng/uL | 122.4 | 110.7 | 137 | 133.5 |
ug in 35 uL | 4.3 | 3.9 | 4.8 | 4.7 |
uL sample | 35 | 35 | 35 | 35 |
uL 10X buffer | 5 | 5 | 5 | 5 |
uL Lambda Exonuclease (5 U/uL) | 3 | 3 | 3 | 3 |
uL nfH2O | 7 | 7 | 7 | 7 |
Total | 50 | 50 | 50 | 50 |
- Incubate for 1 hr at 37C
- Heat inactivate for 10 minutes at 75C
- Purify with Zymo column, elute with 20 uL nfH2O
Nanodrop Results[edit]
Sample | V6S2 | V6S3 |
ng/uL ssDNA | 187.6 | 228.1 |
ug ssDNA | 3.8 | 4.6 |
Yield (Max ~50%) | 42.16 | 48.02 |
Again, since the theoretical maximum of this reaction is 50% (we eliminate 1 strand), this yields an effective 85% and 96% yield from the reaction/column, which is pretty good.