I have been trying to extract DNA from Candida isolates in a 96-well setup by using self-prepared buffers. However, after the lysis step when I am adding potassium acetate to the pellet all I am getting is a precipitate. I can't move ahead with the next step as there is no supernatant. Can anyone please help me with this?
Dear Parul,
It sounds as if you have too my biological material in your sample, and after lysis this is aggregating into a large precipitate when you and the NaOAc. You cloud avoid this by (i) decreasing the amount of yeast you are extracting DNA from; (ii) increasing all of the volumes of buffers you use; or (iii) add more equilibrated buffer (lysis plus NaOAc) and mixing well before centrifuging to remove the pellet.
Regards, Andrew.
Andrew J Spiers - Thank you so much for your suggestions. I am following a protocol that has already been optimized for other yeasts and stated that it would work on Candida too. As you mentioned above, I did play around with the volumes of the buffers but still ended up getting a precipitate. I am attaching herewith the protocol that I am following. Kindly let me know if you have any comments regarding the same. I would really appreciate your help.
Dear Parul,
I've done similar DNA extractions from bacteria - not yeast - but this looks like a sensible protocol to follow.
When things are not working, it is sensible to take the time to try out different things.
In the past (with entirely different assays), I have had a problem with the quality / purity of SDS. So have you done a blank extraction with a no-yeast culture? Use an old plate, and go through things to see if you are not getting a large precipitate resulting from problems with buffers / chemicals.
Can you actually see that your yeast are being lysed? The suspension should go clear, and if you try taking some out with a pipette, it should be very viscous.
I note you precipitate the cell debris and proteins etc. at -20°C for 5 - 10 min. For plasmid preparations form bacterial cultures, I normally do this step at room temperature - so give that a go to see if you get a smaller precipitate.
Spinning at 4,000 rpm seems a bit slow to me - though the relative g-force (xg) is dependent on the rotor you use. Can you spin your plates any faster? When we do this using 1.6 ml centrifuge tubes (Eppendorfs) we spin at max. speed (14,000 rpm). A low centrifugation will result in a less-dense precipitate being formed; higher xg will smear it out on the plate bottom.
Finally, before you abandon this protocol, how about starting off with your overnight culture, and diluting 2x down to (say) 1/2, 1/4, 1/8, 1/16 and 1/32 and doing the extractions on these to see that it is not biomass (or some characteristic of your yeast) that is causing the problem?
Andrew
- Badges
- Science topic
- Similar topics
- Phytochemistry
- Extracts