Team:UCLA/Notebook/Spider Silk Genetics/22 April 2015
4/22/2015
1 Bacterial Transformation
∙ Observed many colonies growing on the plates. We will do colony PCR to verify proper insertion.
2 Colony PCR
∙ Picked four colonies from each plate. Resuspended each each colony in 100 uL water. Used protocol below:
| 1x Reaction (uL) | 12x Reaction (uL) | |
| 5x Q5 buffer | 5 | 60 |
| 10 mM dNTPs | 0.5 | 6 |
| 10 uM For (VF2) | 1.25 | 15 |
| 10 uM Rev (post-elution G-03) | 1.25 | 15 |
| Colony (Resuspended) | 1 uL | 1 uL |
| 5x GC Enhancer | 5 | 60 |
| Q5 polymerase | 0.25 | 3 |
| ddH2O | 10.75 | 129 |
| Total | 25 | 300 |
| 98 C | 3 min |
| 98 C | 10 sec |
| 66 C | 15 sec |
| 72 C | 15 sec |
| repeat from step 2 | 25 x |
| 72 C | 2 min |
| 12 C | hold |
3 Results
∙ We picked colonies 3 and 4 from each construct and grew 5 mL liquid culture for miniprep and sequencing tomorrow. Renumbered the cultures to 1 and 2 respectively.
4 Column purification of MaSp2 PCA
∙ Used zymo kit to purify yesterday’s MaSp2 PCA reactions. Eluted in 10 uL EB. Results below:
| Concentration (ng/uL) | |
| 2AB | 279.34 |
| 2BC | 154.57 |
| 2CA | 236.36 |
5 BsaI Digestion of PCA products from 4/21/2015
∙ Set up 2x 25 uL reactions for each:
| 2AB(279 ng/uL) | 2BC (155 ng/uL) | 2CA (237 ng/uL) | |
| DNA | 2 uL ( 560 ng) | 5 uL ( 770 ng) | 2.5 uL ( 590 ng) |
| 10x Cutsmart | 2.5 uL: | 2.5 uL | 2.5 uL |
| ddH2O | 18.5 uL | 15.5 uL: | 18 uL |
| BsaI | 2 uL | 2 uL | 2 uL |
| Total | 25 uL | 25 uL | 25 uL |
| 50 C | 2 hr |
| 65 C | 20 min |
∙ The digestion products were column purified using zymo kit with the following yields:
| Concentration (ng/uL) | A 260/280 | |
| 2AB | 24.7 | 2.09 |
| 2BC | 55.17 | 1.9 |
| 2CA | 22.38 | 2.14 |