DNA extraction for amplicon sequencing

Vinn · 02-09-2015, 09:56 AM

Hi folks,

Sorry for having a very basic question here. I am going to use amplicon sequencing to study endophytes in tree. I was wondering whether it is necessary to perform DNA extraction under laminar flow cabinet?

I know that it is important to prevent contaminations from airborne spores, especially the primer pair I use is universal one (ITS1F-ITS4). However, it is not practical to perform the extraction under LAF -- not mentioning about fungal spores that would contaminate in liquid nitrogen and etc. Do you have any opinions/suggestions?

Also would it help to reduce contamination and distortion of the results if I run PCR at low number of cycles (e.g. 20) instead of 35?

Thank you very much in advance!

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02-09-2015, 09:56 AM	#1
Vinn Member Location: Sweden Join Date: Nov 2014 Posts: 18	DNA extraction for amplicon sequencing Hi folks, Sorry for having a very basic question here. I am going to use amplicon sequencing to study endophytes in tree. I was wondering whether it is necessary to perform DNA extraction under laminar flow cabinet? I know that it is important to prevent contaminations from airborne spores, especially the primer pair I use is universal one (ITS1F-ITS4). However, it is not practical to perform the extraction under LAF -- not mentioning about fungal spores that would contaminate in liquid nitrogen and etc. Do you have any opinions/suggestions? Also would it help to reduce contamination and distortion of the results if I run PCR at low number of cycles (e.g. 20) instead of 35? Thank you very much in advance!