Help with making a Dimer

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Help with making a Dimer

Postby Kenny17 » Jul 10 2017 7:27 am

Hi, I need urgent help to make a dimer of Maize Streak Virus (MSV) genome cloned in pGEM3zf+ vector. I am currently having issues with how to digest the plasmid such that it will yield MSV genomes fragments from which I can easily ligate a dimer of the genome into my acceptor plasmid for further processing.

Thanks.
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Re: Help with making a Dimer

Postby relaxin » Jul 11 2017 11:38 am

What are the issues? If there are no convenient restriction sites, you can just copy the MSV genome by PCR using primers with built-in restriction site. If the genome is too big, you can copy it in several fragments with unique restriction ends and then ligate them together.
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Re: Help with making a Dimer

Postby Kenny17 » Jul 14 2017 2:05 am

relaxin wrote:What are the issues? If there are no convenient restriction sites, you can just copy the MSV genome by PCR using primers with built-in restriction site. If the genome is too big, you can copy it in several fragments with unique restriction ends and then ligate them together.


Thanks for your response, actually I want to use the BamHI and aatII site on the plasmid as i need an enzyme that can cut the vector backbone but MSV genome which aatII will do. I just need other tips and tricks that can make me work smarter(the vector map is attached). Also, can you please send me the protocol you suggested in your reply earlier?
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Re: Help with making a Dimer

Postby relaxin » Jul 15 2017 10:01 am

If you cut out the MSV genome with BamHI and AatII, you will have an extra 2000 bp from the plasmid backbone. If there is no other convenient restriction site available, you can copy the MSV genome by PCR. It is just regular PCR, with built-in restriction site at the 5'-end of the primers. However, this may be complicated if the MSV genome is big, as you have to copy it in pieces and then splice them together.

How big is the MSV genome?
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Re: Help with making a Dimer

Postby Kenny17 » Jul 18 2017 10:31 am

The MSV genome is approximately 3kb it is quite very small.

Regards
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Re: Help with making a Dimer

Postby relaxin » Jul 19 2017 11:08 am

Then you can copy it in one piece by PCR. You can add restriction site(s) at the 5'-end of the primers to facilitate the subcloning into your desired vector.
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Re: Help with making a Dimer

Postby Kenny17 » Jul 20 2017 3:43 am

Thank you.
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