Hello, for routine work, we are using the QIAamp DNA Mini Kit to isolate the DNA from bacteria. This kit works fine when the intention is to use the DNA for routine PCR. However, the yield is too small for NGS library prep (Nextera XT): We measured between 0.1-0.7 ng/ul . This concentration is even too small to correctly determine the concentration of DNA using Qubit or Quantus. Thus, we either have to optimize our protocol or to establish a new protocol. For example: Option 1:
We may use the Mini Kit and add lysozyme or lysostaphin to lyse the rigid multilayered cell wall of S.aureus and other bacteria.
Option 2:
Establish e.g. the MagAttract HMW DNA Kit from Qiagen with the addition of Lysostaphin
Which kit do you recommend?
Hi Sarah,
When extracting gDNA from Gram Positive bacteria, cells lysis, protein precipitation/degradation and RNA degradation should not be overlooked. Therefore, cell lysis solution (e.g. Lysozyme), protein elimination buffer (e.g. Proteinase K and) and RNase (RNase A) are strongly required to efficiently increase the amount of extracted DNA. Thus, the MasterPure Gram Positive DNA Purification kit is appropriate for the purpose, OR the QIAamp DNA Mini Kit with supplementation of at least Lysozyme, Proteinase K and/or RNase A in the earlier stages of the extraction process.
In addition to NSA's suggestion in order to increase the concentration of the elute, also kindly consider heating (e.g. 65- 70ºC) the buffer (even better use nuclease-free water) to precipitate DNA.
All these put together could help overcome this bottleneck.
Good Luck !
Thank you Nsa. We are using a lot of starting material - thus I think that the problem is that we have never used lysozym or lysostaphin to better lyse the cells.
Hi Sarah,
When extracting gDNA from Gram Positive bacteria, cells lysis, protein precipitation/degradation and RNA degradation should not be overlooked. Therefore, cell lysis solution (e.g. Lysozyme), protein elimination buffer (e.g. Proteinase K and) and RNase (RNase A) are strongly required to efficiently increase the amount of extracted DNA. Thus, the MasterPure Gram Positive DNA Purification kit is appropriate for the purpose, OR the QIAamp DNA Mini Kit with supplementation of at least Lysozyme, Proteinase K and/or RNase A in the earlier stages of the extraction process.
In addition to NSA's suggestion in order to increase the concentration of the elute, also kindly consider heating (e.g. 65- 70ºC) the buffer (even better use nuclease-free water) to precipitate DNA.
All these put together could help overcome this bottleneck.
Good Luck !
We use MP BIOMEDICALS FastDNA SPIN Kit for Soil and you will get enough DNA for NGS. Otherwise you will need to try old school phenol-chloroform extraction.
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