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insert in the reverse orientation after transformation


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5 replies to this topic

#1 jaya2020

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Posted 23 May 2010 - 12:01 PM

hi

I have done cloning into Ecoli using blunt end ligation, after filling the DNA with Klenow. The colonies grew well on agar, but after screening, I found that all had the DNA in the reverse orientation. I screened nearly 50 colonies. Your help will be greatly appreciated.

jaya

#2 pDNA

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Posted 23 May 2010 - 01:23 PM

most of the time one orientation is prefered over the other resulting in a certain ratio after screening ...maybe your insert is toxic or a heavy burden to the cell in the orientation that you have not been able to find during screening.

The only thing i can recommend is to do directional cloning instead of blunt cloning if possible ...this should solve your problem! ...or to screen more colonies!
What plasmid are you using? Is there a bacterial promoter upstream the cloning site ...if so try to use conditions that supress the activity of this promoter when plating your ligation reaction (e.g. add glucose when using an arabinose-promoter for expression and so on).

Regards,
p

#3 jaya2020

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Posted 23 May 2010 - 10:19 PM

most of the time one orientation is prefered over the other resulting in a certain ratio after screening ...maybe your insert is toxic or a heavy burden to the cell in the orientation that you have not been able to find during screening.

The only thing i can recommend is to do directional cloning instead of blunt cloning if possible ...this should solve your problem! ...or to screen more colonies!
What plasmid are you using? Is there a bacterial promoter upstream the cloning site ...if so try to use conditions that supress the activity of this promoter when plating your ligation reaction (e.g. add glucose when using an arabinose-promoter for expression and so on).

Regards,
p


Thank you for the reply. Let me try as you suggested,

regards,

jaya

#4 jaya2020

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Posted 25 May 2010 - 11:02 AM

most of the time one orientation is prefered over the other resulting in a certain ratio after screening ...maybe your insert is toxic or a heavy burden to the cell in the orientation that you have not been able to find during screening.

The only thing i can recommend is to do directional cloning instead of blunt cloning if possible ...this should solve your problem! ...or to screen more colonies!
What plasmid are you using? Is there a bacterial promoter upstream the cloning site ...if so try to use conditions that supress the activity of this promoter when plating your ligation reaction (e.g. add glucose when using an arabinose-promoter for expression and so on).

Regards,
p



hi

Please clarify me this doubt. If my insert is toxic, it means that the protein it is going to encode is toxic to the bacteria, so that it does not grow. If this is the case will directional cloning solve the issue? How will the toxic product be converted to a non toxic protein. Help is greatly appreciated

regards,

jaya

#5 pDNA

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Posted 25 May 2010 - 11:33 AM

if your protein is toxic then directional cloning will not solve your problem.

if this is the case you can either try to produce the protein as inclusion bodies, incorrectly folded or try to minimize the uninduced activity of your bacterial promoter used for expression.

There should be a lot of literature available for the expression of toxic genes.

Regards,
p

#6 jaya2020

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Posted 26 May 2010 - 12:45 PM

if your protein is toxic then directional cloning will not solve your problem.

if this is the case you can either try to produce the protein as inclusion bodies, incorrectly folded or try to minimize the uninduced activity of your bacterial promoter used for expression.

There should be a lot of literature available for the expression of toxic genes.

Regards,
p



Thank you. I will search the literature

regards,

jaya




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