I am using thermofisher midiprep kit for my plasmid isolation. I got 43ng/uL last year. However, I'm using the same protocol now and getting consistently lower yield (around 7ng/uL) in 350 uL EB. Can someone give any advice how to troubleshoot this problem?
I grew back in 1 mL LB media for 8 hrs and then 50 uL from that in transferred into 50 mL LB media for overnight culture.
It could be due to the plasmid you are trying to purify or the bacteria strain.
I recommend:
- Grow your bacteria for longer and to higher density
- Check your shaker incubator condition, make sure your bacterial flask/tube has loose cap and provide oxygen for the cells
- Use less volume of EB (you can always add more water to dilute your DNA)
- Pre-warm your EB or water to 50 before adding to your column
- Add half of your EB to column, incubate in RT for 5 minutes, centrifuge and then add the other half of EB to the same column, incubate and centrifuge in the same collection tube (basically elute twice in the same collection tube to increase the chance of capturing DNA)
Good luck! :)
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