Sequencing - (Jun/24/2006 )

hi all


am new to this forum,and currently working in a lab for the summer as a technician. I have a problem and that is that i am not molecular microbiology based , infact i'm a physiologist.

I have cloned a DNA fragment in the BamHI site of pUC 19 and i dont know what this sequence is..so i have to send it to be sequenced ( in the lab, we deal with genome express)

i checked out their website and they have universal primers, sepecific vector primers etc...pUC 19 exist but i have no idea about designing primers!!! never done it before!! and nobody in the lab is cooperative with me!! im so lost

can anyone please help me out!!

what do i have to do!! as i said its a pUC19 with an insert( sequence of interest, unknown) at its BamHI site

i really appreciate your help, im so lost and my boss is very strict

thankyou all

Hi,

I am not very experienced either, but from what I know if its not wrong, if its a universal primer( as you have said, they have the primer u want) the lab doing the sequencing may already have the primer and will provide for you, so maybe don need to design primers?Just send them the plasmid that u want i to be sequenced?

is it possible to use the pUC19 M13 forward and reverse primers??? i know i cloned my insert in BamHI...what do u guys think, have any of you used these two primers before?


many thanx

pUC19 has sites for all of the M13 forward and reverse primers. Either would probably sequence your insert, depending on how long it is, but if you really need to be sure about the sequence, it is routine to sequence in both directions, with a forward and reverse primer.

I agree with phage. Moreover, if you need to design your own primer(s), I would advice you to check on the website of genome express: they should have guidelines for primer design

i think that i'm just going to use the M13 forward and reverse primers as they are listed as the commercially pUC19 specific primers and the BamHI site is within them. As for the size of my insert, i dont know yet as i will be doing the minipreps of my selected colonies next week. I will digest by BamHI to verify that they have the insert in them!! wish me luck!! hope it works.

thanx alot for the advice

ciao