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
