Daniel:Notebook/HiResChrPaint/2014-7-9

From ZhangLabWiki
Jump to navigation Jump to search

Strand Control (Started 07/08/2014)[edit]

Back to Calendar

DpnII Digest (from 07/08/2014)[edit]

  1. Column Purification, elute with 40 uL
  2. 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

File:2014 07 09 DpnII.jpg

Lambda Exonuclease Digestion[edit]

  1. 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
  1. Incubate for 1 hr at 37C
  2. Heat inactivate for 10 minutes at 75C
  3. 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.