fetal mistake in my hot start PCR - (May/16/2006 )

i have been doing COBRA with hot-start for 1 month, but i only get dimers. my boss try to do troubleshooting for me, so my boss doing the PCR with same reagent, same primer, and the condition, and he got band! then something maybe wrong in my PCR procedure or technique, then my boss mix all the reagents except Template DNA and Taq, then divided into two group, both of us use one of the group respectively. and he got band, i have no band. so this is clear there must be mistake in adding DNA or Taq(hot start). i do not think i will make any mistake in adding DNA, because i am naive to hot start PCR, i think i make mistake in adding Taq manually. (we use manully hot start, add diluted Taq after 95 degree for 2 min)

but I and my boss can not find where is the mistake for me in adding Taq, i do not think this is a complicated procedure, but i fail in this for 1 month! i guess the Taq will float at the top of taq vails heating at 95 degree, so i only pipette water but no Taq in to the PCR vails, if i insert my pipetter deep into the taq vail, or something else?

I fail in the "adding Taq step", is there a mystery?

It's possible your pipette technique is not up to scratch.

I have had a couple of bright students in my current lab, go through the same problems and it was only watching them physically add the reagents to the PCR tube, I found that their pipette handling was a little lousy.

You can always try a "pseudo hot start" something I do all the time, which is to add everything to your PCR tube on ice, close the tubes and then set the PCR machine running, once up to 95C place the PCR tubes into the heat block and away you go. Seems to work well for me, and there is less risk of cross contamination (however this is not an issue for you at the moment).

Good luck!
Nick

hi, nick, you are right
i performed the "psudo hot start PCR", and it works, although there is strong dimer. so i wonder what is the problem in my hot start procedure.

usually, we dilute 5 units Taq into 50 ul water, heated with PCR tube for 95 degree for 2min, add 10 ul diluted taq to each pcr tube.

i guess i can change for the following things, but i am not sure if these will work at all.

1)because the Taq solution volume incrase after heat, when i pipette Taq for 10 ul, i actually take less Taq than 10ul. so i will add a little more. in addition, the Taq (solution with glycerol) may floated to upper when heat, so just before pipette i have to mix them again.
2)when i insert the pipetter into the pcr vial through mineral oil layer, some taq may dissolved into mineral oil.
3)some taq may evaporate, so i have to add quickly.
4) some times i pipette bubbles
5)after adding, pipette for 1 times more for mix.

i will try again

i failed again, after pay attention to the above points.

i did quite well in normal PCR, but i can not with hot start PCR. beside what i mentioned in above post, what is other tips of mannual hot start pcr?