Haynes Lab:Notebook/CRISPR Editing/2015/01/14

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01/14/2015

Picked colonies from 6 of the transformation plates from yesterday into 2-6mL cultures to miniprep tomorrow.

Testing PCR primers
I have a ton of primers that amplify across my target sequence. I tested all of them by pairing all the forward primers with P198 and all the reverse primers with P197, both of which I know work very well.

95 for 3min
30 cycles
95 for 30s
60 for 30s
72 for 2 min
72 for 10min
' F primer R primer exp length
1 P165 P198 1439
2 P167 P198 1406
3 P151 P198 1411
4 P163 P198 1370
5 P153 P198 1329
6 P149 P198 1283
7 P159 P198 1132
8 P173 P198 1087
9 P161 P198 1052
10 P175 P198 968
11 P197 P158 1516
12 P197 P172 1496
13 P197 P156 1388
14 P197 P170 1380
15 P197 P176 811
16 P197 P162 756
17 P197 P174 714
18 P197 P160 624
19 P197 P150 509
20 P197 P198 1660

Results
all had strong single bands except P156. P156 has a band at the correct size but it's faint.