ORF region amplification problem - (Apr/08/2008 )

Hi,

I am trying to amplify orf of my gene from start to stop codon , I limited scope to design a good primer . I tried to design two sets of primer
set 1
FW 5'- ATG CTG CAC AGG CGC TTC AGT GTT CAG AAA GC -3'
rev 5'- TTA GTA CAG GTC CCG CAT GAT CTC C -3'
and Set 2

ORFF2 5' ATG-CTG-CAC-AGG-CGC-TTC 3'

ORFR2 5' GTC-TTT-AGT-ACA-GGT-CCC-GCA-TG 3'

set 1 didnt give me any result, set two gave me very weak band around my product size which is around 1.5 kb.

is it always that difficult to amplify ORF , are there any suggestions

regards
Vani

You will have to give us a lot more information so that we can try and figure out what is going wrong.

PCR can go wrong in SO MANY ways

What is your template DNA?? (eg viral, bacterial, plasmid, etc etc)

Have you tried the following??
- altering your annealing temperature
- titrating your primer concentration
- titrating your template concentration

There really are heaps of things that could be going on.....