Difference between PCR/cloning DNA in plasmids - (Jun/19/2011 )

Hi there,

So I have a couple of questions..the first:

Soo, if the principle of PCR os to amplify a gene of interest (GOI) and thereby produce many copies of the gene what is the difference if I took my gene of interest and inserted it into a plasmid, transformed into e.coli and therefore make many copies of the gene when e.coli divides etc...so my question is...essentially both do the same job right?...make many copies of the GOI...so when would you use one or the other...?

Another question:

Say I have a gene that I'm interested in studying..how do I isolate it...I assume get restriction enzymes, thus making many fragments, use same RE on plasmid and then you can do the whole blue-white selection to see if the gene of interest has been inserted into the MCS...but my question is...say I use BAMH1 and EcoR1 and take chromosomal DNA from the organism I am studying...what happens if there are heaps of genes that are produced from using these 2 enzymes...I mean EcoR1 recognizes the sequence GAATCC--surely that sequence appears numerous times throughout ones' genome...I don't get how you would specifically isolate this gene using this technique of getting the DNA and adding restriction enzymes..

So I guess the other way was if you know the DNA sequence of the GOI, would one normally make primers and add restriction sites each end of primer..therefore you can amplify the gene of interest and the restriction sites will always be at the end of the GOI when each copy is made and then when its inserted into the vector it will bind to the complementary ends created by those same RE's used in the MCS...

Man, I really hope I make sense here..and you see where I'm trying to go with these questions..

Thanks,
b_06er

Hello,

Looks like you are new to molecular biology. Gene amplification by PCR can do many things, isolating/cloning a gene is one of them. Because of amplification, you're able to isolate/single out your GOI from the genome DNA or total RNA. With the restriction enzyme recognition sequence in the primers integrated by PCR, you can clone the GOI to a vector for many purposes.

Both PCR and vector can achive amplification/isolation of genes, but PCR give you a quicker and easier way to isolate and clone a GOI.

Hi silkworm,

Thanks for the reply...as you have said with the RE recognition sequence in the primers integrated by PCR...how does this work essentially..I understand the process of PCR but if you have your sequence "hanging" at the end of the primer will the complementary strand of the sequence just be made during the first cycle...and if so, how exactly..is it carried out by the Taq polymerase? The taq polymerase will extend the 3 end and yet the recognition sequence will be at the end of the 5 end. and every subsequent cycle of pcr will always have the primer binding to the gene, not the complementary recognition sequence...

silkworm on Sun Jun 19 14:40:50 2011 said: