Team:UCLA/Notebook/Spider Silk Genetics/28 August 2015
Contents
8/28/2015
Gel Purification for M1 monomers
- Used zymo kit, eluted in 12 uL of solution.
- Yields were typical, around 25 ng/uL
Sequencing for M1, M2 monomers
- Due to the presence of single bp mutations in the previous sequencing results, we wanted to check the sequence of the monomers to ensure that they are correct.
- Sent each monomer sample for sequencing.
PCR Amplification for M1-12, M1/2[1:2]-12
- 3x 50 uL reactions for each. Used 1 uL of the ICA eluate as template.
Volume (uL) | |
---|---|
5x Q5 Buffer | 10 |
10 mM dNTPs | 1 |
10 uM For (F-03) | 2.5 |
10 uM Rev (G-03) | 2.5 |
Template | 1 |
Q5 Polymerase | 0.5 |
ddH2O | 32.5 |
Total | 50 uL |
98 C | 30 sec |
98 C | 10 sec |
66 C | 20 sec |
72 C | 30 sec |
repeat from step 2 | 20x |
72 C | 2 min |
12 C | hold |
- Visualized on 1% TAE gel with 2 uL of 1 kb ladder.
- Excised the 1324 bp bands for extraction.