...
- The segment to be amplified is the right side of a gene coding for YFP (other people are amplifying the left side)
- The goal will be to amplify the right and left sides of the gene and then assemble them together into a functional gene
- Diluted the two primer solutions (forward primer and reverse)
- Added pre-made "PCR Mix" and primers to DNA
- Set PCR machine to the cycle specifications called for in the protocol
3/14/14 & 3/16/14
Practiced Gibson Assembly
Gibson Reaction Mix for All Samples
Mass (μg) | Enzyme (μL) | Buffer (μL) | Total (μL) |
|
1 | 1 | 2 | 20 |
|
|
|
|
|
|
| Con (ng/μL) | Vol DNA (μL) | Vol H2O (μL) |
|
1.1 | 151 | 6.62 | 10.38 | CMU-Nde1 |
1.2 | 267 | 3.75 | 13.25 |
|
1.3 | 141 | 7.09 | 9.91 |
|
2.1 | 652 | 1.53 | 15.47 | TRE-t-Sta1 |
2.2 | 263 | 3.80 | 13.20 |
|
2.3 | 212 | 4.72 | 12.28 |
|
4.1 | 149 | 6.71 | 10.29 | TRE-t-Sta1 |
4.2 | 415 | 2.41 | 14.59 |
|
4.3 | 583 | 1.72 | 15.28 |
|
6.1 | 353 | 2.83 | 14.17 | TRE-t-Sta1 |
6.2 | 593 | 1.69 | 15.31 |
|
6.3 | 361 | 2.77 | 14.23 |
|
7.1 | 448 | 2.23 | 14.77 | Het1a-Xhol |
7.2 | 237 | 4.22 | 12.78 |
|
7.3 | 186 | 5.38 | 11.62 |
|
8.1 | 246 | 4.07 | 12.93 | Het1a-Lac-BamH1 |
8.2 | 182 | 5.49 | 11.51 |
|
8.3 | 871 | 1.15 | 15.85 |
|
9.1 | 410 | 2.44 | 14.56 | TRE-t-Sta1 |
9.2 | 214 | 4.67 | 12.33 |
|
9.3 | 185 | 5.41 | 11.59 |
|
10.1 | 285 | 3.51 | 13.49 | CMU-Nde1 |
10.2 | 547 | 1.83 | 15.17 |
|
10.3 | 388 | 2.58 | 14.42 |
|
PCR reaction to check assemblies; ran the products through two gels (one gel's purpose was to check the products' size by inspection under UV, the other gel's purpose was to extract the products)
Extracted plasmids from transformed bacteria (LR reaction); measured plasmid concentration with nanodrop