Team:UCLA/Notebook/Spider Silk Genetics/1 June 2015
Contents
6/1/2015
- We are preparing to test the initiators, terminators and caps whenever they arrive.
VF/R amplification of MaSp 2AB, 2BC, 2CA
- We followed the same protocol as on 5/27/2015.
| 2AB (112 pg/uL) | 2BC (205 pg/uL) | 2CA (219 pg/uL) | |
|---|---|---|---|
| 5x Q5 Buffer | 81 uL | ||
| 10 mM dNTPs | 8.1 uL | ||
| 10 uM For | 22 uL | ||
| 10 uM Rev | 22 uL | ||
| Template | 8 uL | 4.3 uL | 4 uL |
| 5x GC Enhancer | 81 uL | ||
| Q5 Polymerase | 4.05 uL | ||
| ddH2O | 178.9 uL | 182.6 uL | 182.9 uL |
| Total | 405 uL | ||
| 98 C | 30 sec |
| 98 C | 10 sec |
| 63 C | 15 sec |
| 72 C | 15 sec |
| repeat from step 2 | 18x |
| 72 C | 2 min |
| 12 C | hold |
PCR Purification
- Used Zymo DNA clean and concentrator-5 to purify the above PCR.
- Split each 8-strip in two and purified separately, then eluted using the same 10 uL of ddH2O.
| Concentration (ng/uL) | A 260/280 | |
|---|---|---|
| 2AB | 194.10 | 1.86 |
| 2BC | 379.37 | 1.71 |
| 2CA | 318.99 | 1.70 |
BsaI Digestion
- Digested 1.5 ug of each PCR product from above in 50 uL with 4 uL of BsaI.
- Same protocol as 5/28/2015.
| AB (194.1 ng/uL) | BC (379.4 ng/uL) | CA (319.0 ng/uL) | |
|---|---|---|---|
| BsaI | 4 uL | ||
| 10x Cutsmart | 5 uL | ||
| DNA | 7.7 uL | 4.0 uL | 4.7 uL |
| ddH2O | 33.3 uL | 37.0 uL | 36.3 uL |
| Total | 50 uL | ||
Results
- Cast 250 mL of 1.8% TAE gel to visualize digestion results. We used 2 uL of NEB 50 bp ladder.
- It appears that digestion this time is more complete than previously.
- We excised the 102 bp band for gel recovery tomorrow.