Team:UCLA/Notebook/Spider Silk Genetics/2 July 2015
Contents
7/2/2015
Gel Extraction for Yesterday
- Extracted BsaI digested MaSp, as well as the 6-mer.
| Concentration (ng/uL) | A 260/280 | |
|---|---|---|
| AB-1 | 14.19 | 2.52 |
| AB-2 | 24.4 | 3.42 |
| BC-1 | 22.21 | 2.97 |
| BC-2 | 25.99 | 2.88 |
| CA-1 | 32.92 | 3.74 |
| CA-2 | 26.98 | 3.03 |
| 6-mer (no beads) | 42.64 | 3.75 |
PCR Amplification of 6-mer (no beads)
- Used post elution primers to amplify the extracted 6-mer.
- Will cycle test to determine optimal conditions.
- Set up 2x 25 uL reactions, testing 17x and 20x cycles.
- For template, used 100 uL of 1:100 dilution of the gel-extracted 6-mer (no beads). ~43 pg of template per reaction.
| Volume (uL) | |
|---|---|
| 5x Q5 buffer | 5 |
| 10 mM dNTPs | 0.5 |
| 10 uM For | 1.25 |
| 10 uM Rev | 1.25 |
| Template (~43 pg) | 1 |
| Q5 Polymerase | 0.25 |
| 5x GC enhancer | 5 |
| ddH2O | 10.75 |
| Total | 25 uL |
| 98 C | 30 sec |
| 98 C | 10 sec |
| 66 C | 15 sec |
| 72 C | 29 sec |
| repeat from step 2 | 17x, 20x |
| 72 C | 2 min |
| 12 C | hold |
Results
- Cast 1% TAE gel, used 2 uL of 100 bp ladder to visualize result.
- This PCR confirms that the 712 bp band is the desired 6 mer.
- The presence of other amplification products may be due to contamination of template by 3-mers (406 bp size) or IT construct (100 bp size). May also be due to non-specific amplification.
- We can try temperature testing in the future.
- I also want to try PCR without the GC-enhancer.
Gel-Extraction
- Excised the 712 band from above, and used qiagen kit to extract.
- Got 17.9 ng/uL, A 260/280: 2.5