Bands appear poor/not at all and do not appear consistantly & primer bands a - (Jul/24/2006 )

Hi there hope some of you guys could help me with these PCR problems im having - im new to this.
Trying to amplify 127bp interferon gamma gene. The first pcr i did on this worked perfectly. But since then its not worked well at all. The bands do not appear clearly and when they do some bands appear, some dont. Then i repeat with same DNA samples and ones that appeared before do not the next time and vice versa!

I did not change the reagents between the first successful pcr and the subsequent unsuccessful pcrs. But i have since changed reagents done another pcr in a bid to identify the source of the problem.

Ive altered the concentration of the DNA, this usually results in a total absence of any bands.

The protocol and reagents have worked previoulsy when this has been done before. The DNA is apparently ok cos working for other peoples work (so im told!)

Ive ran the pcr products on a 3% metaphor agarose gel. I run 18 samples at a time and the products should all be at the same point along the gel (L-R) obviously, but there seems to be a "wave" along the band>! but i think this is a separate problem.

I have checked everything and am at a loss! If anyone wants to ask about what ive done then post im usually at the computer waiting patiently for ANOTHER pcr run to finish!!! Thanks PQ

hmmmm

if I were you, and I know this sounds a little goofy, but believe me it could prove to be VERY helpful...get someone else who knows PCR to watch you do everything...and I mean everything...for the set-up of your next run. I would have picked a contaminated reagent or a problem with the template integrity, except your co-workers can get it to work. this is not good
is there anything that you are using that your co-workers are not, that you can point a finger at? otherwise, there may be something in your technique that is amiss

and please don't feel bad, my intention is not to pick on you...but PCR can require a lot of finesse sometimes and it can be difficult to pick up initially

also, something is fishy with your gels and it may be helpful to sort that out...are there differences in the way you do gels as opposed to your co-workers? I do not know 'metaphor agarose' so can't help there, but smiling or frowning bands are usually bad juju and point to mechanical or pH / running issues...

good luck

hmmmm

if I were you, and I know this sounds a little goofy, but believe me it could prove to be VERY helpful...get someone else who knows PCR to watch you do everything...and I mean everything...for the set-up of your next run. I would have picked a contaminated reagent or a problem with the template integrity, except your co-workers can get it to work. this is not good
is there anything that you are using that your co-workers are not, that you can point a finger at? otherwise, there may be something in your technique that is amiss

and please don't feel bad, my intention is not to pick on you...but PCR can require a lot of finesse sometimes and it can be difficult to pick up initially

also, something is fishy with your gels and it may be helpful to sort that out...are there differences in the way you do gels as opposed to your co-workers? I do not know 'metaphor agarose' so can't help there, but smiling or frowning bands are usually bad juju and point to mechanical or pH / running issues...

good luck