Need advice on PCR Workstation - laminar flow or not?

[urchin]

We are starting up a project and plan to use MiSeq for analysis later. I am purchasing some new equipment and can't find any specifics on why it might be better to get a PCR workstation with laminar flow rather than dead air!! We have a regular fume hood in the lab already. I just want to decrease possible contamination for PCR prep and planned to get a UV PCR workstation, not realizing this was a possible issue. In the past I have only done projects with low risk of contamination, but now will be using universal primers.

Please help me with pros and cons of each type if you have done these type of projects before.

[HESmith]

For typical library preps, laminar flow is not necessary. A decontaminated, dedicated work bench and instrument, with clean reagents/consumables and good technique (most important!), is sufficient.

For single-cell/extremely-low-input samples, follow the recommended guidelines for your chosen method of library prep.

[bunce]

Hi Urchin, In my experience of 'minimising' contamination/artefacts the workflow (as opposed to the equipment) is the thing to get right - The best advice I could give you here is to:
1) Separate out pre-and post PCR
2) Be careful about input DNA copy-number you seed the reaction with
3) Be careful about the index tag combinations you use (or re-use)
We published a paper recently on amplicon sequencing workflow - might be of use;

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0124671

good luck with your research.

[urchin]

Thanks, I'll get the paper ASAP. We really do want to reduce our possible problems from the outset. I already know things will go wrong, it's inevitable, but still...

[bunce]

Hi Urchin - drop me an email if you want to discuss any of this. Contamination in NGS workflows (especially involving amplicons) is, as you point out, something you want to get right from the outset. The work we have done on ancient DNA, forensic and biosecurity means that we have given this topic much thought! Cheers.