protein expression - Preparation of starter cultures (Sep/16/2009 )
Hello everyone,
Im trying to express a his tagged protein in E.coli BL21 DE3. I prepare the starter culture by inoculation of a single colony of the clone im 50 ml LB . I leave it overnight, next morning i use it to inoculate another set of LB media. Does any one know how long the starter culture should be grown?. i ve seen literature that recommends growth of starter cultures upto 16 hrs. Pls help
-chn09-
chn09 on Sep 16 2009, 12:30 PM said:
Hello everyone,
Im trying to express a his tagged protein in E.coli BL21 DE3. I prepare the starter culture by inoculation of a single colony of the clone im 50 ml LB . I leave it overnight, next morning i use it to inoculate another set of LB media. Does any one know how long the starter culture should be grown?. i ve seen literature that recommends growth of starter cultures upto 16 hrs. Pls help
Im trying to express a his tagged protein in E.coli BL21 DE3. I prepare the starter culture by inoculation of a single colony of the clone im 50 ml LB . I leave it overnight, next morning i use it to inoculate another set of LB media. Does any one know how long the starter culture should be grown?. i ve seen literature that recommends growth of starter cultures upto 16 hrs. Pls help
Hi,
Typically you can grow the starter culture for about 16-20 hrs.
And may be inoculate about 1/10th volume of the medium, next day...!!
-KAUSHIK THAKKAR-
chn09 on Sep 16 2009, 02:00 PM said:
Hello everyone,
Im trying to express a his tagged protein in E.coli BL21 DE3. I prepare the starter culture by inoculation of a single colony of the clone im 50 ml LB . I leave it overnight, next morning i use it to inoculate another set of LB media. Does any one know how long the starter culture should be grown?. i ve seen literature that recommends growth of starter cultures upto 16 hrs. Pls help
Im trying to express a his tagged protein in E.coli BL21 DE3. I prepare the starter culture by inoculation of a single colony of the clone im 50 ml LB . I leave it overnight, next morning i use it to inoculate another set of LB media. Does any one know how long the starter culture should be grown?. i ve seen literature that recommends growth of starter cultures upto 16 hrs. Pls help
I usually inoculate 10ml culture in a 50ml tube overnight(approxiamately 18 hours), next day transfer 5ml into 100ml prewarmed media.
-Wolfgang-
KAUSHIK THAKKAR on Sep 16 2009, 12:55 AM said:
chn09 on Sep 16 2009, 12:30 PM said:
Hello everyone,
Im trying to express a his tagged protein in E.coli BL21 DE3. I prepare the starter culture by inoculation of a single colony of the clone im 50 ml LB . I leave it overnight, next morning i use it to inoculate another set of LB media. Does any one know how long the starter culture should be grown?. i ve seen literature that recommends growth of starter cultures upto 16 hrs. Pls help
Im trying to express a his tagged protein in E.coli BL21 DE3. I prepare the starter culture by inoculation of a single colony of the clone im 50 ml LB . I leave it overnight, next morning i use it to inoculate another set of LB media. Does any one know how long the starter culture should be grown?. i ve seen literature that recommends growth of starter cultures upto 16 hrs. Pls help
Hi,
Typically you can grow the starter culture for about 16-20 hrs.
And may be inoculate about 1/10th volume of the medium, next day...!!
This is related to my another post...why to use only 1/10th volume of starter culture? Instead can't it be grown for less time and the whole starter culture used? If the starter culture is grown for 16-20 hrs, wont it cross the log phase (OD 0.6)?
-ram-
ram on Nov 4 2009, 10:55 AM said:
KAUSHIK THAKKAR on Sep 16 2009, 12:55 AM said:
chn09 on Sep 16 2009, 12:30 PM said:
Hello everyone,
Im trying to express a his tagged protein in E.coli BL21 DE3. I prepare the starter culture by inoculation of a single colony of the clone im 50 ml LB . I leave it overnight, next morning i use it to inoculate another set of LB media. Does any one know how long the starter culture should be grown?. i ve seen literature that recommends growth of starter cultures upto 16 hrs. Pls help
Im trying to express a his tagged protein in E.coli BL21 DE3. I prepare the starter culture by inoculation of a single colony of the clone im 50 ml LB . I leave it overnight, next morning i use it to inoculate another set of LB media. Does any one know how long the starter culture should be grown?. i ve seen literature that recommends growth of starter cultures upto 16 hrs. Pls help
Hi,
Typically you can grow the starter culture for about 16-20 hrs.
And may be inoculate about 1/10th volume of the medium, next day...!!
This is related to my another post...why to use only 1/10th volume of starter culture? Instead can't it be grown for less time and the whole starter culture used? If the starter culture is grown for 16-20 hrs, wont it cross the log phase (OD 0.6)?
I grow my starter culture of 50 mL for about 12 hrs. and then use the whole for incoulating a 1L medium....
-DRN-
DRN on Nov 3 2009, 11:41 PM said:
ram on Nov 4 2009, 10:55 AM said:
KAUSHIK THAKKAR on Sep 16 2009, 12:55 AM said:
chn09 on Sep 16 2009, 12:30 PM said:
Hello everyone,
Im trying to express a his tagged protein in E.coli BL21 DE3. I prepare the starter culture by inoculation of a single colony of the clone im 50 ml LB . I leave it overnight, next morning i use it to inoculate another set of LB media. Does any one know how long the starter culture should be grown?. i ve seen literature that recommends growth of starter cultures upto 16 hrs. Pls help
Im trying to express a his tagged protein in E.coli BL21 DE3. I prepare the starter culture by inoculation of a single colony of the clone im 50 ml LB . I leave it overnight, next morning i use it to inoculate another set of LB media. Does any one know how long the starter culture should be grown?. i ve seen literature that recommends growth of starter cultures upto 16 hrs. Pls help
Hi,
Typically you can grow the starter culture for about 16-20 hrs.
And may be inoculate about 1/10th volume of the medium, next day...!!
This is related to my another post...why to use only 1/10th volume of starter culture? Instead can't it be grown for less time and the whole starter culture used? If the starter culture is grown for 16-20 hrs, wont it cross the log phase (OD 0.6)?
I grow my starter culture of 50 mL for about 12 hrs. and then use the whole for incoulating a 1L medium....
Hi DRN
Did u anytime take OD of your 12 hrs grown starter culture...Did u get proper expression after doing so? If yes, can u just post a snapshot of SDS-PAGE having your sample and negative control loaded on the same gel?
-ram-
ram on Nov 4 2009, 03:06 PM said:
DRN on Nov 3 2009, 11:41 PM said:
ram on Nov 4 2009, 10:55 AM said:
KAUSHIK THAKKAR on Sep 16 2009, 12:55 AM said:
chn09 on Sep 16 2009, 12:30 PM said:
Hello everyone,
Im trying to express a his tagged protein in E.coli BL21 DE3. I prepare the starter culture by inoculation of a single colony of the clone im 50 ml LB . I leave it overnight, next morning i use it to inoculate another set of LB media. Does any one know how long the starter culture should be grown?. i ve seen literature that recommends growth of starter cultures upto 16 hrs. Pls help
Im trying to express a his tagged protein in E.coli BL21 DE3. I prepare the starter culture by inoculation of a single colony of the clone im 50 ml LB . I leave it overnight, next morning i use it to inoculate another set of LB media. Does any one know how long the starter culture should be grown?. i ve seen literature that recommends growth of starter cultures upto 16 hrs. Pls help
Hi,
Typically you can grow the starter culture for about 16-20 hrs.
And may be inoculate about 1/10th volume of the medium, next day...!!
This is related to my another post...why to use only 1/10th volume of starter culture? Instead can't it be grown for less time and the whole starter culture used? If the starter culture is grown for 16-20 hrs, wont it cross the log phase (OD 0.6)?
I grow my starter culture of 50 mL for about 12 hrs. and then use the whole for incoulating a 1L medium....
Hi DRN
Did u anytime take OD of your 12 hrs grown starter culture...Did u get proper expression after doing so? If yes, can u just post a snapshot of SDS-PAGE having your sample and negative control loaded on the same gel?
Hi Ram
I never take the od...
.......12 hrs. is an approximate time, sometimes i keep it for more time (too lazy 2 come early in the morning).........i mean its kind of routine stuff, so i don't bother with taking the od. And yes, no matter, whether i keep for 12 hrs. or 14 hrs. (in fact, i hv also kept the culture just for 7 hrs.), I do get protein expression....As for the gel pic, i don't hv any pics with the negative control . Simply because, i would hv already checked the expression on mini-scale with the negative control. Hope this helps.... -DRN-
Thanks for the info DRN!
nice to know that u get nice expression at such variable time of expression which vector r u using by the way?
Right now I am using Invitrogen pCR T7 TOPO TA Expression Kit. And I am not able to see any difference between SDS-PAGE patterns of induced and uninduced sample! 
-ram-
ram on Nov 4 2009, 09:51 PM said:
Thanks for the info DRN!
nice to know that u get nice expression at such variable time of expression which vector r u using by the way?
Right now I am using Invitrogen pCR T7 TOPO TA Expression Kit. And I am not able to see any difference between SDS-PAGE patterns of induced and uninduced sample!
nice to know that u get nice expression at such variable time of expression
which vector r u using by the way?Right now I am using Invitrogen pCR T7 TOPO TA Expression Kit. And I am not able to see any difference between SDS-PAGE patterns of induced and uninduced sample!
Hi Ram
I use pET vectors , mostly pET 21 ©....I haven't used the TOPO TA expression kit, though.....If you are not able to see the difference, it could also be because of leaky expression....is there any difference b/w ur empty vector (induced) and clone (induced)????
-DRN-
DRN on Nov 4 2009, 10:30 PM said:
ram on Nov 4 2009, 09:51 PM said:
Thanks for the info DRN!
nice to know that u get nice expression at such variable time of expression which vector r u using by the way?
Right now I am using Invitrogen pCR T7 TOPO TA Expression Kit. And I am not able to see any difference between SDS-PAGE patterns of induced and uninduced sample!
nice to know that u get nice expression at such variable time of expression
which vector r u using by the way?Right now I am using Invitrogen pCR T7 TOPO TA Expression Kit. And I am not able to see any difference between SDS-PAGE patterns of induced and uninduced sample!
Hi Ram
I use pET vectors , mostly pET 21 ©....I haven't used the TOPO TA expression kit, though.....If you are not able to see the difference, it could also be because of leaky expression....is there any difference b/w ur empty vector (induced) and clone (induced)????
My negative control was the uninduced sample aliquoted just before induction at 0.6 OD. Would there be leaky expression at this point also?
I had thought of doing control expression with an empty vector ...but as u know its a costly TOPO kit n i have the smallest one with only 10 reaction...so I cannot give away 1 only for empty expression...is there any other way?
-ram-