Haynes Lab:Notebook/CRISPR Editing/2015/03/24

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03/24/2015

Ran on gel and did PCR purification kit for unt and g025 treated luc14 and gal4+dox cekks, nested PCR with P173/215.

Surveyor hybridization protocol on 200ng in 15ul

Cell type Treatment Rep 260 280 260/280 ng/µL ul for 200ng ul hyb buff ul water
Luc14 Unt 1 0.037 0.019 1.89 36.604 5.46 1.50 8.04
Luc14 Unt 2 0.025 0.014 1.745 25.137 7.96 1.50 5.54
Luc14 Unt 3 0.024 0.013 1.792 23.704 8.44 1.50 5.06
Gal4-EED+dox Unt 1 0.027 0.015 1.79 26.773 7.47 1.50 6.03
Gal4-EED+dox Unt 2 0.026 0.014 1.879 26.1 7.66 1.50 5.84
Gal4-EED+dox Unt 3 0.022 0.012 1.874 21.554 9.28 1.50 4.22
Luc14 g025 1 0.023 0.012 1.897 23.003 8.69 1.50 4.81
Luc14 g025 2 0.024 0.013 1.763 23.653 8.46 1.50 5.04
Luc14 g025 3 0.023 0.014 1.649 22.935 8.72 1.50 4.78
Gal4-EED+dox g025 1 0.023 0.013 1.76 23.111 8.65 1.50 4.85
Gal4-EED+dox g025 2 0.018 0.01 1.765 17.939 11.15 1.50 2.35
Gal4-EED+dox g025 3 0.026 0.016 1.59 26.08 7.67 1.50 5.83


Cut with 1ul Surveyor enzyme and 1ul enhancer. Ran on BA.


PCR purified

Cell type Treatment rep 260 280 260/280 ng/µL ul for 200ng ul of hyb buff ul water
Luc14 Unt 1 0.037 0.019 1.922 36.604 5.46 1.50 8.04
Luc14 Unt 2 0.03 0.016 1.84 30.185 6.63 1.50 6.87
Luc14 Unt 3 0.028 0.015 1.845 27.725 7.21 1.50 6.29
Gal4-EED+dox Unt 1 0.033 0.019 1.699 32.525 6.15 1.50 7.35
Gal4-EED+dox Unt 2 0.032 0.019 1.674 31.888 6.27 1.50 7.23
Gal4-EED+dox Unt 3 0.028 0.015 1.83 27.876 7.17 1.50 6.33
Luc14 g044 1 0.028 0.015 1.818 28.126 7.11 1.50 6.39
Luc14 g044 2 0.031 0.017 1.753 30.514 6.55 1.50 6.95
Luc14 g044 3 0.03 0.017 1.732 30.199 6.62 1.50 6.88
Gal4-EED+dox g044 1 0.025 0.014 1.766 25.351 7.89 1.50 5.61
Gal4-EED+dox g044 2 0.032 0.017 1.81 31.522 6.34 1.50 7.16
Gal4-EED+dox g044 3 0.034 0.02 1.696 33.517 5.97 1.50 7.53
Luc14 g048 1 0.026 0.016 1.675 26.453 7.56 1.50 5.94
Luc14 g048 2 0.018 0.011 1.648 18.227 10.97 1.50 2.53
Luc14 g048 3 0.023 0.012 1.921 22.727 8.80 1.50 4.70
Gal4-EED+dox g048 1 0.027 0.015 1.795 26.675 7.50 1.50 6.00
Gal4-EED+dox g048 2 0.022 0.013 1.742 22.397 8.93 1.50 4.57
Gal4-EED+dox g048 3 0.028 0.017 1.673 27.608 7.24 1.50 6.26

g023 and g046 gDNA extraction

Cell type Treatment rep 260 280 260/280 ng/µL ul for 200ng
Luc14 g023 1 0.127 0.064 1.983 127.048 1.57
Luc14 g023 2 0.107 0.055 1.948 106.752 1.87
Luc14 g023 3 0.067 0.034 1.966 67.196 2.98
Gal4-EED +dox g023 1 0.096 0.047 2.051 95.9 2.09
Gal4-EED +dox g023 2 0.103 0.05 2.067 103.347 1.94
Gal4-EED +dox g023 3 0.097 0.05 1.95 97.202 2.06
Luc14 g046 1 0.151 0.077 1.97 151.192 1.32
Luc14 g046 2 0.098 0.049 1.992 98.095 2.04
Luc14 g046 3 0.093 0.047 1.969 93.4 2.14
Gal4-EED +dox g046 1 0.135 0.069 1.959 135.41 1.48
Gal4-EED +dox g046 2 0.086 0.043 2.002 86.375 2.32
Gal4-EED +dox g046 3 0.08 0.042 1.922 79.87 2.50