emPCR question

[dasenkom]

Hi, I'm new to 454 sequencing and in the process of setting up our lab to perform GS Junior runs. Our FAS recommended that we have a PCR hood for setting up the emPCR. Is this necessary? Do most labs have PCR hoods for this step? Do the benefits of having the PCR hood for emPCR setup outweigh the costs of purchasing one?
Thanks,
Mark

[mohdsos]

I use a normal hood no air flow only light. and it's fine .

[Jacky76]

You will take some advantage for preventing any possible contaminations by using PCR enclosure. Roche people recommend us to divide the spaces for pre-PCR from post-PCR to prevent cross contanmination. But it will be less necessary to have two rooms if you have PCR enclosure because it can replace pre-PCR. There are two major steps that contamination causative which are Nebulization and emulsion breaking. DNA molecules can fly during this jobs, so it also is recommended that you to have two ventilation hood systems for the both steps.

[ajthomas]

It is rather important to not mix the space or equipment used for the pre- and post-emPCR steps. The strands you remove during the emPCR breaking/recovery/enrichment process are excellent templates for emPCR, and there are a lot of those molecules compared to what you start with. Getting even a little on a pipette or something and then using that same equipment/lab space for setting up the emPCR is just asking for contamination. Yes, you need a hood or a separate room and a dedicated set of pipettes and other equipment for setting up your emPCR.

[SeqAA]

It may not seem like a big deal at first, but you will better off starting in a hood.

[anujgupta]

All depend upon your hands-on expertise. Emulsion breaking specially in LV results in aerosol formation that's why this area is termed as dirty. It should be separated from that used for emulsion preparation. I agree with SeqAA, better starting with a hood.

[MBS]

Better use a hood

You'll find out soon, that a hood is good, when you sequence different organisms, because you will find the alien sequences in the following run. This can be quite embarrassing if the data will go out to some other group.

You probably never find out, if you only work on one or closely related organisms, because the alien reads are hard to find. They may just spoil your results.

Use UV light and bleach in it! Same is for the library preps!