4/28 PCR to prepare 2 honeybee constructs for cloning
We are repeating the same PCR from 4/26, but only at one temperature instead of a gradient, and we are scaling the reaction up to 50 ul.
PCR recipe
Component |
Volume |
5X Q5 Reaction Buffer |
10 |
10 mM dNTPs |
1.0 |
10 uM Forward (primer 3/7)
|
2.5 |
10 uM Reverse (primer 8)
|
2.5 |
Template (diluted to 1ng/uL) |
1.0 |
Q5 High Fidelity DNA Polymerase
|
0.50 |
Nuclease Free Water |
32.5 |
PCR program (using benchling annealing temperatures)
Temperature (C) |
Time (Min:sec) |
98 |
0:30 |
98 x 30 cycles |
0:10 |
grad.
62 (silk) 58.4 (silk+prom) x 30
|
0:20 |
72 x 30
|
0:30 |
72(diluted to 1ng/uL) |
2:00 |
10 |
hold |
PCR products run on 1% agarose gel.
There were some non specific bands, but the appropriate bands were present. However, the bands were kind of off. I think this is due to the samples running weird on the gel. B/c the first two lanes were the exact same sample, just aliquoted, and they ran fairly differently.
Performed a gel extraction and purified using Qiagen min elute gel extraction kit
Results : 115 ng/ ul for silk tube #1, 86 ng/ul tube #2 (8.8 ul each tube)
136 ng/ul promoter + silk