Team:UCLA/Notebook/Spider Silk Genetics/21 July 2015
Contents
7/21/2015
Colony Results
- Results of yesterday's transformations.
- Glycerol plates all have colonies.
- M2-9(T7) has very small colonies.
- M2-12(1C3) has no colonies.
- Will redo PCR, ligation, transformation.
PCA of MaSp1 AB, BC, CA
- Scaled up the protocol from yesterday for 50 uL reactions for MaSp1 AB, BC, CA each.
- We used 64 C as the annealing temperature.
PCR of 12-mer
- Starting from PCR of 12-mer due to transformation failure.
- We could re-transform with the leftover ligation, but we are not certain if it is good.
- Repeated protocol from 7/16/2015.
- Scaled to 1x 50 uL reaction.
Results of MaSp1 PCA, 12-mer PCR
- Cast 1.5% TAE gel to visualize, used 2 uL of 100 bp ladder.
- Ran 5 uL of the MaSp1 PCR to verify presence of desired product.
- Results indicate that the PCA of MaSp1 was successful.
- Results indicate that PCR of the M2-12-mer was also successful.
- Excised the bands corresponding to the M2-12-mer.
- Gel-purified: 20.38 ng/uL
Digestion of MaSp1 AB, BC, CA and M2-12-mer for cloning
- Digest with EcoRI, PstI for cloning into pSB1C3.
- Also digested some linearized backbone.
Backbone (25 ng/uL) | M1-AB (179.98 ng/uL) | M1-BC (262.32 ng/uL) | M1-CA (181.57 ng/uuL) | |
---|---|---|---|---|
EcoRI | 1 uL | 1 uL | 1 uL | 1 uL |
PstI | 1 uL | 1 uL | 1 uL | 1 uL |
10x 2.1 NEBuffer | 5 uL | 5 uL | 5 uL | 5 uL |
DNA (~1 ug) | 24 uL | 5.6 uL | 3.8 uL | 5.5 uL |
ddH2O | 19 uL | 37.4 uL | 39.2 uL | 37.5 uL |
Total | 50 uL | 50 uL | 50 uL | 50 uL |
- Digest 37 C 1.5 hrs, heat kill 65 C 20 min.
- Post-digestion: PCR clean-up using Zymo Clean and Concentrator.
Ligation
- Ligate M1 AB, BC, CA and M2-12(E,P digest) into pSB1C3(E,P digest).
M-12 (20.38 ng/uL) | M1-AB (36.82 ng/uL) | M1-BC (27.82 ng/uL) | M1-CA (37.53 ng/uL) | |
---|---|---|---|---|
10x T4 Ligase Buffer | 2 uL | 2 uL | 2 uL | 2 uL |
pSB1C3 (50 ng) | 1.4 uL | 1.4 uL | 1.4 uL | 1.4 uL |
Insert | 4.4 uL | 1.4 uL | 1.8 uL | 1.3 uL |
ddH20 | 11.2 uL | 14.2 uL | 13.8 uL | 14.3 uL |
Total | 20 uuL | 20 uL | 29 uL | 20 uL |
- Ligate 25 C for 25 min, heat kill 65 C 10 min.
Transformation
- Transform M2-12(1C3), M1-AB, BC, CA into electrocompetent DH5(alpha) cells.
- Arc times:
- M2-12(1C3): 5.4
- M1-AB: 5.6
- M1-BC: 5.6
- M1-CA: 5.6
- Plated all the cells.