6 replies to this topic

[nanobio]

Hi,

Very recently i started investigating in a laboratory, i began woking
with a plasmid pcDNA3-FLAG-p53.
Some days ago i had the need to know the plasmid full sequence. Although
i contacted the company they didn't had it. I've already search google for it but
came empty handed
A friend said send me an article where the plasmid insertions are described, and said
that with that information he thinks i could know the full sequence.

Article Link

My real problem is that i don't know how to do it. Could someone please
kindly help me.

Thanks in advance

[bob1]

If you know the plasmid (pcDNA3 or pcDNA3-flag) then you can sequence the 5' and 3' ends of the insert (flag-p53 or p53) and surrounding plasmid. This will tell you the sequence where the insert has been cloned. Assume that the rest of the plasmid has the same sequence as the parent plasmid, and that the rest of the p53 sequence is correct (you could sequence it all if you want) - get these sequences from the web (e.g. company or from Genbank etc.), and align/contig sequences with the sequences you got from your sequencing...

Voila, you have the full sequence.

[laurequillo]

Just use The T7 primer and the Sp6 primer, it should give you the sequence.

Or you just can start with the T7 primer, and start your sequencing only in one direction. Then once you have your first sequencing (les say about 1000 base) then you use this sequence and create another primer....but that is the "long" way. It is easier to use the T7 primer and the Sp6 primer to amplify your gene of interest!!

[nanobio]

thank you for your help, but i stand with a doubt by this method is
possible to have the full sequence without the need to sequence
the plasmid in the lab right?

[bob1]

It is possible, but only if you know where the p53 was cloned in (i.e. what restriction enzymes used, or T/A cloning?), you would also need to know if there were spacers between the flag and the p53, extra stop codons added etc...

[nanobio]

bob1, on Feb 11 2010, 03:16 PM, said:

It is possible, but only if you know where the p53 was cloned in (i.e. what restriction enzymes used, or T/A cloning?), you would also need to know if there were spacers between the flag and the p53, extra stop codons added etc...

Thanks a lot for the help bob1 it was quite enlighning, i'm going to search for that information.

[laurequillo]

bob1, on Feb 12 2010, 12:16 AM, said:

It is possible, but only if you know where the p53 was cloned in (i.e. what restriction enzymes used, or T/A cloning?), you would also need to know if there were spacers between the flag and the p53, extra stop codons added etc...

Of course if you have all that information (if the flag is N or C terminal, the kodak sequence, the sites used...) you dont need to sequence it. I was thinking that it was to confirm that the plasmid was ok, without mutations or something strange, my mistake.

Just remember, if you are gonna send the plasmid to sequence remember to leave a space of 50-90 bases between the primer and the sequence you want to get

Edited by laurequillo, 11 February 2010 - 10:56 PM.