12/19/2014
Sample Read#
|
260
|
280
|
260/280
|
ng/µL
|
ul for 400ng
|
g023 |
0.105 |
0.055 |
1.916 |
105.241 |
3.80
|
g025 |
0.087 |
0.048 |
1.837 |
87.358 |
4.58
|
g029 |
0.089 |
0.049 |
1.841 |
89.293 |
4.48
|
g030 |
0.108 |
0.058 |
1.87 |
107.618 |
3.72
|
g032 |
0.117 |
0.063 |
1.841 |
116.542 |
3.43
|
untreated |
0.164 |
0.088 |
1.873 |
164.02 |
2.44
|
Sample Read#
|
260
|
280
|
260/280
|
ng/µL
|
400ng
|
h2o
|
g033 |
0.12 |
0.063 |
1.908 |
120.29 |
3.33 |
5.67
|
g034 |
0.114 |
0.061 |
1.866 |
114.301 |
3.50 |
5.50
|
g036 |
0.125 |
0.067 |
1.86 |
125.052 |
3.20 |
5.80
|
g039 |
0.101 |
0.055 |
1.85 |
101.372 |
3.95 |
5.05
|
g040 |
0.128 |
0.069 |
1.86 |
128.165 |
3.12 |
5.88
|
g041 |
0.126 |
0.068 |
1.861 |
126.349 |
3.17 |
5.83
|
g042 |
0.179 |
0.095 |
1.876 |
179.179 |
2.23 |
6.77
|
g043 |
0.076 |
0.042 |
1.833 |
76.202 |
5.25 |
3.75
|
Column purified all but 31, 35, 38. Ran PCR again for those ones, this time I raised the annealing temp to 66. It still gave a product but I think there might still be off target bands.
Ran 10ul annealing reactions, 400ng for untreated (three times), 23, 25, 29, 30, 32, 33, 34, 36, 39, 40, 41, and 42. Cut with 1ul surveyor, 1ul enhancer, at 42deg C for 45 minutes, nuclease negative samples I added 2ul elution solution to. Added 1.5ul stop solution. Ran some on bioanalyzer in biodesign, 1:20 dilutions, 1ul each.
All samples without nuclease had a pretty band at 1660bp, all samples with nuclease, including untreated Luc14, were completely digested, no longer saw the 1660 peak, only what would be a smear on a gel.
|