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- 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
File:2014 07 09 DpnII.jpg
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.