Difference between revisions of "Part:BBa K1228002"
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== RESULT == | == RESULT == | ||
| − | 1. The recombinant strains which were trans formed with the plasmid pht-304 were inoculated into 5 ml LB broth containing 25μg erythromycin ml− 1, shaken at 37° C overnight, and then 100 ml LB medium supplemented with erythromycin was inoculated with 1 ml recombinant B.subtilis overnight culture. The cultures were grown at 37°C with shaking at 200 rpm for 10h, | + | 1.The recombinant strains which were trans formed with the plasmid pht-304 were inoculated into 5 ml LB broth containing 25μg erythromycin ml− 1, shaken at 37° C overnight, and then 100 ml LB medium supplemented with erythromycin was inoculated with 1 ml recombinant B.subtilis overnight culture. The cultures were grown at 37°C with shaking at 200 rpm for 10h, |
| − | 2. The strains carrying only the pht-304 shuttle vector with no insert were used as controls. | + | |
| − | 3. The liquid culture was centrifuged at 10000× g for 10 min , and the cell-free supernatant was then concentrated with Millipore. | + | 2.The strains carrying only the pht-304 shuttle vector with no insert were used as controls. |
| − | 4. Add 20 ul 5XSDS sample buffer to 80 ul concentrated culture and heat 95°C for 10 minutes; | + | |
| + | 3.The liquid culture was centrifuged at 10000× g for 10 min , and the cell-free supernatant was then concentrated with Millipore. | ||
| + | 4.Add 20 ul 5XSDS sample buffer to 80 ul concentrated culture and heat 95°C for 10 minutes; | ||
5. Load 20 μl onto 12% SDS-PAGE gel，80V for stacking gel , 120V for separation gel . | 5. Load 20 μl onto 12% SDS-PAGE gel，80V for stacking gel , 120V for separation gel . | ||
6. Make the gel for transfer in transfer buffer: 12V overnight, on ice. | 6. Make the gel for transfer in transfer buffer: 12V overnight, on ice. | ||
Revision as of 03:21, 27 September 2013
Rabies virus strain ERA glycoprotein coding sequence
The RNA genome of the virus encodes five genes whose order is highly conserved. These genes code for nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G) and the viral RNA polymerase (L).The complete genome sequences range from 11,615 to 11,966 nt in length.”Because of containing the neutralizing epitopes which are the targets of vaccine-induced immunity, the glycoprotein can stimulate the organism to produce antibody against Rabies.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 510
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1338
Illegal BsaI site found at 1357
Illegal BsaI.rc site found at 1564
RESULT
1.The recombinant strains which were trans formed with the plasmid pht-304 were inoculated into 5 ml LB broth containing 25μg erythromycin ml− 1, shaken at 37° C overnight, and then 100 ml LB medium supplemented with erythromycin was inoculated with 1 ml recombinant B.subtilis overnight culture. The cultures were grown at 37°C with shaking at 200 rpm for 10h,
2.The strains carrying only the pht-304 shuttle vector with no insert were used as controls.
3.The liquid culture was centrifuged at 10000× g for 10 min , and the cell-free supernatant was then concentrated with Millipore. 4.Add 20 ul 5XSDS sample buffer to 80 ul concentrated culture and heat 95°C for 10 minutes; 5. Load 20 μl onto 12% SDS-PAGE gel，80V for stacking gel , 120V for separation gel . 6. Make the gel for transfer in transfer buffer: 12V overnight, on ice. 7. Block the filter with blocking buffer for 1 hour at room temperature with gentle agitation on a platform shaker. 8. Discard blocking solution and immediately incubate filter with primary antibody. 9. Add 0.005 ml of primary antibody (1:5000) in to blocking solution. Incubate 2 hour at room temperature. with gentle agitation on a platform shaker. 10. Discard blocking solution and wash filter 3 times (5 minutes each time) with TBST. 11. Immediately incubate the filter with secondary antibody, add 0.003ml of secondary antibody solution (1:3000). 12. Incubate 1 – 2 hours at room temperature with gentle agitation. 13. Discard blocking solution and wash filter 3 times (5 minutes each time) with TBST. 14. Chemiluminescence for 1minute.
