Previously, I try to isolate DNA of Neisseria gonorrhoeae by DNA isolation kit but it's quite hard because colonies of it can't be suspend easily in buffer. With vortex and scrape, it's still tough to be dissolve. I will use DNA for PCR and sequencing.
I don't know the boiling assay will be appropriate for these methods. Have anyone had an idea to solve this problem?
Thanks a lot.
HI Natakorn,
I can tell you the procedure that I use for the colonies.
First of all, scrap the colony into a lysis buffer eNAT (from Copan) or any other lysis buffer available to you. For maximum yield, subject your samples to a freeze-thaw cycle of -80°C overnight followed by 95°C for 15 minutes the next day. From this point, you can use any isolation method available to you.
Good luck,
Ali
We have had a similar issues when working with Neisseria meningitidis. Having never worked with gonorrhoeae, I cannot guarantee that all of our practices are relevant to you however, it does sound like a familiar problem.
For whole genome sequencing, we use Qiagen DNeasy kits for the DNA extraction. We have had issues in the past in breaking down the colonies in the lysis buffer (buffer AL and proteinase K). First of all we use a loop to collect a clump of colonies. We then remove the intact clump into the lysis buffer. It is important that the colonies are removed from the loop as quickly as possible to prevent DNA from adhering to the loop. Spinning the loop vigorously between the finger and thumb should help to remove the clump of cells. The cells are then left in the buffer preferably overnight @ 56degC (but at least 2 hours @ 56degC with regular vortexing may be sufficient). The suspension should be transluscent once all the cells are lysed.
I hope this helps.
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