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E coli culture cannot grow overnight.


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#1 widebamboo

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Posted 13 June 2014 - 06:37 AM

I transformed three plasmids into Top 10 (without F') selected by Amp, Tet and Cm. The colonies sometimes grow good overnight as normal sometimes grow really small and needs longer time. but when I start the culture from the colonies, it has problems. They always grow very slow, the overnight culture look transparent like nothing inside, but when I leave them in the room temperature one more day, they will give cloudy culture. I used this cloudy culture to start the larger culture and then induce by IPTG. I got the products which catalyzed by the proteins produced by the three plasmids.

 

also I store the cloudy culture into -80, and then I start them using fresh LB without any antibiotics, they still not grow overnight and need like one more day.

 

anyone has any suggestion? I can grow them normally? Thanks.  



#2 Bio-Lad

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Posted 13 June 2014 - 05:36 PM

Are the genes you express toxic to the cells? Top10 cells don't require induction by IPTG as they lack the LacZ repressor. Maybe try in another strain like DH5a?

I'd be careful about leaving incubating to long without adding fresh antibiotics, though. Some degrade pretty quickly and reduce selection for the plasmid.

Just my thoughts.

Try pLOT, the a free plasmid mapping tool!

http://plasmidplotter.blogspot.com/


#3 Bio-Lad

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Posted 13 June 2014 - 06:11 PM

(Or maybe the concentration of one of the antibiotics is to high? Is one of these a BAC?)

Try pLOT, the a free plasmid mapping tool!

http://plasmidplotter.blogspot.com/


#4 lyok

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Posted 14 June 2014 - 03:13 AM

Are the genes you express toxic to the cells? Top10 cells don't require induction by IPTG as they lack the LacZ repressor. Maybe try in another strain like DH5a?

I'd be careful about leaving incubating to long without adding fresh antibiotics, though. Some degrade pretty quickly and reduce selection for the plasmid.

Just my thoughts.

 

Do you have an idea on how long you could incubate them?

 

I sometimes incubate >12hours and I noticed my minipreps were not that good (very low yield). I wonder its because of this reason.

The OD I reach is +- 2 (seems low , not?)

 

THe cells are grown in 3ml LB + antibiotic
 


Edited by lyok, 14 June 2014 - 03:13 AM.


#5 Bio-Lad

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Posted 14 June 2014 - 11:47 AM

I guess it depends.  Can you say which plasmids you are using, what the LacZ-promoter-driven product  is and which concentration you are using for of each antibiotic?


Try pLOT, the a free plasmid mapping tool!

http://plasmidplotter.blogspot.com/


#6 lyok

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Posted 15 June 2014 - 04:01 AM

I guess it depends.  Can you say which plasmids you are using, what the LacZ-promoter-driven product  is and which concentration you are using for of each antibiotic?

 

I used amp (100µg/ml). The plasmids: its not some sort of commercial plasmid. So cant really give some general details about it.

 

I dont use a LacZ promoter.

 

The other plasmid is kanamycine selection based. (50µg/ml)



#7 Bio-Lad

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Posted 15 June 2014 - 02:04 PM

Ah, if you are using IPTG to induce, would I be correct in thinking you have some sort of RNA polymerase driven by the LacZ promoter and your genes of interest transcribed by that RNA pol?

 

Oh, I didn't see anything about Kan in your original post.  You had said you selected with Amp, Cam, Tet.

 

If you can't give out too many details, can you at least say whether these are high or low-copy plasmids?  If you use very low copy plasmids, you may need to reduce the concentration of Tet and Cam.  For BACs, for example, we use 6.25ug/mL Cam and 12ug/mL Tet.  

 

If the copy number is low, it could explain why your cultures initially don't grow.  I could see two things happening, either:

  1) They don't grow until such a time as the Cam/Tet have degraded enough to allow them to go or

  2) The chloramphenicol could lead to a plasmid amplification type off effect until there are enough copies of the resistance genes to overcome the antibiotics.

 

 Without any more information as to what kind of construct you are using, this is my best guess =\  

I'd try lowering the Tet/Cam to the above concentrations and seeing if that results in better growth overnight.

 

Let me know if that helps.


Edited by Bio-Lad, 15 June 2014 - 02:07 PM.

Try pLOT, the a free plasmid mapping tool!

http://plasmidplotter.blogspot.com/


#8 Bio-Lad

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Posted 15 June 2014 - 03:43 PM

One last thought. These plasmids, have you checked to make sure that the origins of replication aren't in the same compatibility group?


Edited by Bio-Lad, 15 June 2014 - 06:12 PM.

Try pLOT, the a free plasmid mapping tool!

http://plasmidplotter.blogspot.com/


#9 lyok

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Posted 15 June 2014 - 09:30 PM

I am a different person than the topicstarter, he used IPTG, me I dont.

 

I will have to check whether its high or low copy, but I think its high copy. You need to origin of replication to know its high or low copy?

And in the same group of compatibility? Its 1 plasmid per sample (the antibiotic selection is in different tubes , its different plasmids, they are seperated, not in 1 e.coli cell)

 

BTW: an OD of 2 after overnight growth, is this not good enough than? (that you think they dont grow enough).


Edited by lyok, 15 June 2014 - 09:31 PM.


#10 Bio-Lad

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Posted 16 June 2014 - 02:57 AM

Oh! Ha ha. Here I thought I was still taking to the OP! Sorry about that! I've been sleep deprived lately due to our baby waking us up all night so I'm going to blame her for this! I was getting really confused. Lol.

What kind of total yield are you getting and what protocol are you using to get your plasmid, Ivok?

Edited by Bio-Lad, 16 June 2014 - 04:20 AM.

Try pLOT, the a free plasmid mapping tool!

http://plasmidplotter.blogspot.com/


#11 lyok

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Posted 17 June 2014 - 12:33 PM

Oh! Ha ha. Here I thought I was still taking to the OP! Sorry about that! I've been sleep deprived lately due to our baby waking us up all night so I'm going to blame her for this! I was getting really confused. Lol.

What kind of total yield are you getting and what protocol are you using to get your plasmid, Ivok?

 

I get between 40 and 70 ng/µl. THis is too low (I think?).

 

I am using a sigma aldrich miniprep kit, just a commercial kit.

 

 



#12 Bio-Lad

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Posted 17 June 2014 - 08:01 PM

 

Oh! Ha ha. Here I thought I was still taking to the OP! Sorry about that! I've been sleep deprived lately due to our baby waking us up all night so I'm going to blame her for this! I was getting really confused. Lol.

What kind of total yield are you getting and what protocol are you using to get your plasmid, Ivok?

 

I get between 40 and 70 ng/µl. THis is too low (I think?).

 

I am using a sigma aldrich miniprep kit, just a commercial kit.

 

 

 

 

If eluted with 50ul (promega kit recommendation), that means you got 2-3.5ug total which actually isn't all that bad if you prepped only 1mL of that culture.  It's lower than what I'd expect if you prepped all 3mL.  If this is the case, I'd make sure you are getting complete lysis.  Occasionally if I have a big pellet, I will increase the amount of each buffer by 1.5-2x (resuspension, lysis, neutralization).  This usually helps with incomplete lysis.  I'd try that first if you haven't already.


Try pLOT, the a free plasmid mapping tool!

http://plasmidplotter.blogspot.com/





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