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protein expression - Preparation of starter cultures (Sep/15/2009 )

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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



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


I usually inoculate 10ml culture in a 50ml tube overnight(approxiamately 18 hours), next day transfer 5ml into 100ml prewarmed media. :rolleyes:

-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



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



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



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



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! :D :)

-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 :lol: 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! :lol: :lol:



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! :( :o



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-
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