I am doing a project for the evaluation of MinION-sequencing pipelines regarding MRSA and non-MRSA in terms of identification and resistance gene detection from cultured clinical isolates. My question is if anyone has a suggestion as to which extraction kit/protocol to use for extracting S. aureus DNA successfully without fragmenting/damaging the DNA too much in the process?
Hi Kristoffer,
Extraction using the QIAAMP Tissue or DNA kit (QIAGEN) using the EZ1 system works well for Gram Positive cocci. You might have to incubate the bacteria colonies with Lyzozyme/Lysostaphin, initially for a better DNA yield.
You can extract the DNA using HiPurA™ Bacterial Genomic DNA Purification Kit (Himedia). It has got different protocols for DNA extraction of Gram-positive and Gram-negative bacteria. Make sure you check the quality of your DNA prior to sequencing using Agarose gel electrophoresis. Additionally, any of the A260/A280 ratio below 1.7 would not give you good quality reads. To my knowledge, DNA with an A260/A280 ratio between 1.8-1.9 gives you excellent results. You can quantify it using a Qubit fluorometer for better accuracy. Also, I would recommend not to fragment your DNA prior to sequencing as it would affect the read length.
Hope this answer helps you.
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