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Project Description
Place holder: Quick Blurb on the CHlH project goes here
The 4166 bp ChlH gene was engineered synthetically by Integrated DNA Technologies (IDT) into 3 gblocks:
| 1(G13) |
1678 bp |
| 2 (P2) |
980 bp |
| 3 (3-6) |
1508 bp |
Thursday 6th August 2015
- Attempted assembly of ChlH fragments into CAM and KAN backbones via Gibson Assembly
- Amplified GA mix to check whether gblocks had successfully been assembled. 3 sets of primers were used (table 1).
- Ran amplicons on a 1% agarose gel with GelRed. If assembly was successful, a band around 4.2 kbp is expected.
Table 1: Primers used for PCR
| 1. |
Vector forward (BBF) |
Vector reverse (BBR) |
| 2. |
ChlH forward |
ChlH reverse |
| 3. |
Vector forward 2 (BBVF2) |
Vector reverse 2 (BBVR2) |
- Transformed 2 uL KAN and CAM assembly products into E. coli cells and plated 100 uL and 150 uL of transformants out on nutrient agar plates with appropriate KAN or CAM antibiotic
- Colonies present on all plates (table 2) but transformation efficiency was very low
- Lots of pink colonies - insert not present
Table 2: Colony counts of Gibson Assembly transformants
| |
100 uL plates |
150 uL plates |
Total |
|
pink |
white |
pink |
white |
|
| CAM |
6 |
8 |
3 |
3 |
20 |
| KAN |
1 |
1 |
0 |
1 |
3 |
| Control |
0 |
0 |
5 |
7 |
12 |
Wednesday 12th August 2015
- Harvested white colonies and cultured them in LB broth with the appropriate antibiotics
- 1 white KAN colony
- 5 white CAM colonies
Thursday 13th August 2015
- Isolated plasmids from liquid cultures via GenElute Plasmid Miniprep Kit (Sigma-Aldrich). Quality and concentration of isolated DNA was assessed via NanoDrop and 1% agarose gel stained with GelRed (table 3).
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Table 3: Concentration and purity of isolate plasmid DNA obtained from NanoDrop.
|
Concentration (ng/uL) |
260/280 |
| CAM 1 |
155.3 |
1.95 |
| CAM 2 |
302.1 |
1.91 |
| CAM 3 |
253.5 |
1.94 |
| CAM 4 |
277.8 |
1.95 |
| CAM 5 |
119.5 |
1.85 |
| KAN 1 |
87.2 |
1.91 |
- Performed double restriction digests with EcoRI and EcoRI + PstI, and ran product on a gel to determine if ChlH had been successfully assembled by last weeks Gibson Assembly.
Thursday 27th August 2015
- 260/280 values indicate isolated
- Performed double restriction digests with EcoRI and EcoRI + PstI, and ran product on a gel to determine if ChlH had been successfully assembled by last weeks Gibson Assembly.
- Results of gel indicated single bands for the double digest, suggesting that the restriction digest was unsuccessful. The EcoRI restriction site may have disappeared.
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