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Old 11-19-2014, 08:09 AM   #13
ZAAB
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Location: Boston

Join Date: Apr 2011
Posts: 18
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Seems to me that one of the biggest 'deterrents' of contamination between samples within one prep, between preps across time, etc is to use enough template DNA so that any contamination won't be preferentially amplified.

Our platform may be more-ammenable to this type of prep, as we know our distal ends of the DNA to be sequenced…

We use a 2-stage PCR to build out our Illumina ends. As suggested earlier, the primary PCR uses library-specific oligos, but with about 1/2 of the Illumina adapters extending off the ends.
The Secondary PCR primers introduce the P5/P7 ends, as well as the barcode, but have no complementarity to the initial library, only the distal ends of the primary PCR product that are part of the illumina adapter just inbound of the barcodes.

We perform a PCR clean-up between each step to clean out the primers…
We use 10ng of input DNA into a 50ul PCR reaction, and that amount of template is a ton more than any contamination event unless you're an idiot...
Also, in-line PCR negative controls are always run…
And we don't ever sequence with the same barcodes two runs in a row...
And I am planning on installing the Bleach wash, but we typically only see about 1% of the total yield of DNA going into the 'undetermined' fastQ file.

I got fancy once, and added 1/1000th the amount of primary PCR primers for 2 cycles, and then added in the typical amount of secondary primer (barcode, P5P7)…that works but I don't like opening fresh PCR products and adding more primers to do more PCR. now that's asking for contamination.
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