Haynes Lab:Notebook/CRISPR Editing/2014/12/19

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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.