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blunt end cloning


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#1 deespike

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Posted 02 May 2010 - 12:02 AM

Hi all

I have been trying this blunt end cloning for sometime but had failure all the times. I am trying to ligate a 2.3kb inset into a 13kb vector . I had dephosphorylated this vector , added 18% PEG 6000, but nothing seems to work.

Moreover, sometimes I get colonies from which when I try to isolate plasmid I get always genomic DNA fragment at around 22kb region when compared to the marker. I get these colonies only when I use this 13 kb vector and not in case of other vectors that I have used so far for other cloning purposes. I donotunderstand the appearance of these colonies.

Has anyone had similar problem like that of mine?

Plz help me in this context.

Thanks in advance.

#2 HomeBrew

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Posted 02 May 2010 - 03:47 AM

Are you linearizing the plasmid DNA isolated from your colonies before comparing it to your linear marker on the gel?

#3 deespike

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Posted 02 May 2010 - 04:59 AM

Are you linearizing the plasmid DNA isolated from your colonies before comparing it to your linear marker on the gel?


Yes i try to linearize them but I get smear indicating that it mght be the genomic dna fragment but I donot understand how these appear for this vector only.

When i load the uncut vector in the gel along with the marker it comes as a linear band at around 22kb.

What can i make out of this? I have also tried cloning of 1kb insert into a 12kb vector , a sticky end ligation that worked but i didnt get this kind of confusing result which i am getting with this vector.Its not just once but several times , same thing has happened

Plz help me with this.

Thanks

#4 phage434

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Posted 02 May 2010 - 05:51 AM

You could be getting a plasmid dimer (13K x 2 could look like 22K). Some plasmid origins will replicate with two origins. Since you are having success with cohesive end cloning, why are you trying to do blunt end cloning in such a large plasmid?

#5 deespike

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Posted 02 May 2010 - 08:05 AM

You could be getting a plasmid dimer (13K x 2 could look like 22K). Some plasmid origins will replicate with two origins. Since you are having success with cohesive end cloning, why are you trying to do blunt end cloning in such a large plasmid?

that can be apossibilty but then it should come linearized at 13k but it does not? But it comes somewhat at the same place 22k and with smear also.
Stick ligation is not possible in this case, so im stuck up with this blunt ligation. I use 22C fro blunt is it okay or i should go lower?


Thanks for your suggestion.




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