...
- 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):
| Lane | Segment | Person |
|---|---|---|
| leftmost lane | ladder | n/a |
| 1 | Adapter | KL |
| 2 | Q1-Q9 | AS |
| 3 | Q9-QX | AL |
| 4 | ? | KL |
| 5 | ? | JA |
Extracted plasmids from transformed bacteria (LR reaction); measured plasmid concentration with nanodrop