Help with making a Dimer

Go to the profile of kenny17
Jul 10, 2017
0
6

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.

6 Comments

Go to the profile of relaxin
relaxin 5 months ago

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.

Go to the profile of kenny17
kenny17 5 months ago

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?

Go to the profile of relaxin
relaxin 5 months ago

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?

Go to the profile of kenny17
kenny17 5 months ago

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

Regards

Go to the profile of relaxin
relaxin 5 months ago

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.

Go to the profile of kenny17
kenny17 5 months ago

Thank you.