Difference between revisions of "Part:BBa K1228002"

(→‎RESULT)
(→‎RESULT)
Line 19: Line 19:
  
 
== 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,
 +
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.
 +
 
https://static.igem.org/mediawiki/igem.org/9/96/G-P--Western_Blot.png
 
https://static.igem.org/mediawiki/igem.org/9/96/G-P--Western_Blot.png

Revision as of 02:14, 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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 510
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE 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.

G-P--Western_Blot.png