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Team:Glasgow/LOVresults
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<br>Reverse primer for site directed mutagenesis of LOV2: 5'-CACCAATGAAGTACTGAAGACCGCCTTTGCG-3' </br></br> | <br>Reverse primer for site directed mutagenesis of LOV2: 5'-CACCAATGAAGTACTGAAGACCGCCTTTGCG-3' </br></br> | ||
| - | 5)Streaked LOV2 containing Top10 from glycerol stocks onto 1mM IPTG and Ampicillan agar plates. Kept in dark for 3 hours at 30 degrees. Used Blak Ray lamp, and transilluminator on plates to visualise any fluorescence. No fluorescence was seen. | + | <br>5)Streaked LOV2 containing Top10 from glycerol stocks onto 1mM IPTG and Ampicillan agar plates. Kept in dark for 3 hours at 30 degrees. Used Blak Ray lamp, and transilluminator on plates to visualise any fluorescence. No fluorescence was seen. </br></br> |
| - | Made up liquid cultures of Top10 + LOV2 in 2ml of LB + 2 microlitres Ampicillan. Liquid cultures grown for 4 hours and then 0.5 ml plated onto plates with varying concentrations of IPTG and with 1mM Ampicillan. These were then wrapped in tinfoil and allowed to grow in the 30 degree incubator for 24 hours. They were tested for fluorescence using both the Blak Ray lamp and the transilluminator with riboflavin as a positive control. No fluorescence was seen. | + | <br>Made up liquid cultures of Top10 + LOV2 in 2ml of LB + 2 microlitres Ampicillan. Liquid cultures grown for 4 hours and then 0.5 ml plated onto plates with varying concentrations of IPTG and with 1mM Ampicillan. These were then wrapped in tinfoil and allowed to grow in the 30 degree incubator for 24 hours. They were tested for fluorescence using both the Blak Ray lamp and the transilluminator with riboflavin as a positive control. No fluorescence was seen. </br></br> |
<br>1) | <br>1) | ||
Revision as of 15:04, 21 September 2011
LOV2 Results
Aims
1)Perform restriction digest to ensure we have LOV2 domain. 1) Transform LOV2 domain into Top 10 2) Make glycerol stocks of LOV2 containing Top 10
3) Design site directed mutagenesis primers to get rid of illegal pst1 site
4)To perform site directed mutagenesis on LOV2
5)Make LOV2 fluoresce under UV light.
6)Ligate LOV2 into sumbission vector
7)Submit LOV 2
Methods
The sequence for LOV2 contains an illegal pst1 site3)Forward primer for site-directed mutagenesis of LOV2: 5'-CGCAAAGGCGGTCTTCAGTACTTCATTGGTG-3'
Reverse primer for site directed mutagenesis of LOV2: 5'-CACCAATGAAGTACTGAAGACCGCCTTTGCG-3'
5)Streaked LOV2 containing Top10 from glycerol stocks onto 1mM IPTG and Ampicillan agar plates. Kept in dark for 3 hours at 30 degrees. Used Blak Ray lamp, and transilluminator on plates to visualise any fluorescence. No fluorescence was seen.
Made up liquid cultures of Top10 + LOV2 in 2ml of LB + 2 microlitres Ampicillan. Liquid cultures grown for 4 hours and then 0.5 ml plated onto plates with varying concentrations of IPTG and with 1mM Ampicillan. These were then wrapped in tinfoil and allowed to grow in the 30 degree incubator for 24 hours. They were tested for fluorescence using both the Blak Ray lamp and the transilluminator with riboflavin as a positive control. No fluorescence was seen.
1)
iLOV Results
Aims
1) Design iLOV construct to be synthesised
2) Get iLOV synthesised
3)Transform iLOV cells and make fluoresce
4)Ligate iLOV into sybmission vector
5)Submit iLOV
