Hi i've carried PCR out but not all the lanes have band patterns...can anyone help???

Answer 1

It depends on a few things:
1) Were all the PCRs done using the same primers? If so, then two things might be wrong:
a) Your reactions aren't getting all of what they need to make a product - consider being more careful setting up reactions, or use a master mix technique: in the PCR tubes, add the DNA, in a larger tube add everything else that is needed for all the reactions (minus the DNA of course). Then aliquot this to the tubes containing the DNA. This helps ensure that all of the components of the PCR are present in a much more homogenous mixture. If you do this and you still get variable results, then:
b) The template DNA you used might be damaged or impure. If you can do a master mix reaction and you still get PCRs that don't work, your DNA might be sheared by excessive freeze/thawing, or it might have contaminants in it from the purification process. If the first, then there is nothing you can do but try to obtain a replacement isolation for the DNA. If the latter, then you can try re-precipitating the DNA.

2) If the reactions you made up used different primers, have you used these primers before? if not, then:
a) the primers might be ill-suited for a PCR. Check the Tm and make sure the two primers have Tm's within one degree of each other.
b) Are you using the right annealing temperature? Usually a rule of thumb is to use a temp four degrees below the Tm, but you can experiment to get the optimal temp.
c) is the Tm for the primers unusually high (due to high GC content?). If so, consider using an additive like DMSO to your reaction mix. This chemically forces the primers to act like they have a lower Tm.

3) If you have used these primers successfully before and they are not working now:
a) try mixing the primers well before adding them to the reaction mix. I have found that freshly-thawed primers tend to be more concentrated on the bottom, and it is easy to get the wrong concentration into the reaction mix
b) consider ordering a fresh order of the same primers. If the primers have been sitting a long while in water (or even a tris buffer), they may have degraded, and this would cause them not to work.

As you can see, there are many different possibilities, but the main ones revolve around primers, DNA, and how you mix it. If you have tried all this and still have problems, ask again and I will try to help.

Answer 2

Wally pretty much covered it, but I think it's worthwhile saying that you should also hot start your reactions, and check that you've closed the lid properly. It's a bit 'hand wavey science', but if you put the PCR tubes in the centre of the grid it seems to work better. I suspect, though, if you follow wally's instructions you'll be fine :)

Answer 3

yes.. probably your supervisor.. it depends on a lot of factors that you haven't included in this question that your supervisor would know