How to eliminate unexpected primer dimer during preparation of the gDNA for NGS analysis?

Hello everyone!

Recently, I performed CRISPR activation (human SAM library) screening and harvested genomic DNA. However, when I performed PCR and purified amplified NGS library as described. I found there were unexpected primer dimers.

I wonder if I could use Gel Extraction Kit to eliminate primer dimers (just like step 35 during Amplification of pooled sgRNA library )? Will there be disturbance for NGS after gel extraction of PCR products? Or should I perform PCR again until there are no primer dimers?

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Jade Nagaover 1 year ago

More details of PCR

genomic DNA 250 ng

NGS-Lib-Fwd-1~10, 10 μM 2.5 μL (10 individual reactions)

NGS-Lib-SAM-Rev-5, 10 μM 2.5 μL
KAPA HiFi HotStart Mix, 2X 25 μL

ddH2O to 50 μL

I performed 25 cycles, pooled PCR products and purified them. I acquired interested DNA fragments (>250bp, ~270-280bp), but also observed unexpected bands around 100bp.

Sienna Sphinxover 1 year ago

You could do gel purification, but may lose a lot of DNA. An alternative would be bead-baaed purification such as Ampure XP.

I have used these in the past with great success. You can remove small dimers very effectively with minimal loss on your amplicon

Jade Nagaover 1 year ago

I would follow your advice.

Fuchsia Kelpieover 1 year ago

Adding my thoughts here: agree that Ampure XP is higher recovery, but I usually have excess amplicon for sequencing and I like gel extraction because it's more precise than Ampure

Jade Nagaover 1 year ago

Very helpful!

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