is Microseal B necessary for MiSeq library prep?

[helico93561]

Hi All,

We had been following the MiSeq guideline and finally it's time to restock the microseal B film. Please forgive my ignorance, what is the purpose of microseal B? cant we use some other much cheaper PCR seal?

[brianchase]

Hi Helico,

I believe the purpose of the Microseal B is that is a non-adhesive based seal but still is able to protect the sample from evaporation/cross contamination while heating or cycling. When you remove the seal post sample processing it comes off really easily and uniformly. Regular seals have adhesive and removing them may lead to contamination.

[DRYTCYV]

You mean Microseal® 'A' Film ?? I dont trust in this sealer

[brianchase]

Oops, thanks for the correction, I was thinking about microseal A. Yeah, it doesn't seem very trustworthy but so far it has worked well for us.
Back to OP's questions. I've always used microseal B when called for in the protocol but I don't see why a less expensive option wouldn't work the same. Maybe illumina is getting kickbacks for mentioning BR products in its protocol?

[Ann235]

sealers

I've worked in biotech for 20 + years. The BioRad B sealer is probably the "sticky-est" sealer I've ever used. It's important for the vortexing steps: if any liquid gets up on the seal rim, a lot of sealers will leak. Not good with valuable DNA that presumably will be a problem if it cross contaminates with the adjacent well (in 96 format).

As to the BioRad A sealer, it's been around for years. It was really useful with the older style thermocyclers that had a screw-down heated lid, eg the MJs and PE 9600. You didn't have to worry about whether the lid was too loose or too tight. It is really easy to remove at the end as well. It's a cheap disposable alternative to a reusable, but potentially contaminated, silicon sealer mat.