2 replies to this topic

[DNA]

Hi all,

I have a gene subcloned in to pcDm8 and now i want to insert it in to pcDNA3.
Please tell me the different steps involved in this process. No body has done it in my lab before as they were using it in pcDM8
but i am not happy with it and want to change the vector.

I would appreciate this help.

[molgen]

Although it sounds suspiciously like a home work question I'll give it the benefit of the doubt.

Go to the pcDm8 mapand look at it (this one is with out your insert).
Then look at the pcDNA3 map.

Chose the restriction enzymes that flank your gene.
Cut the gene out.
Clean the fragment (on a gel or column).
Cut the new plasmid with the same restriction enzymes that you cut before.
Ligate it into the new plasmid.

[NemomeN]

Hi DNA, responded to the same question in your other post but for the sake of completeness, I'll copy it here too.

The easiest way would be to find the sequence of your gene, design primers to amplify it and clone it into pcDNA3 with the appropriate restriction enzymes. Ideally, two diffent ones that do not cut into your gene. This is probably the easiest way in theory to do it unless you have the map of your vector that contains the restriction sites in which case the other option above from molgen is good too.