Hello
Used the Qiagen miniprep to look for plasmids in transformed bacteria. I want to confirm I have the plasmid before going to wholesale production. I have too high an A260/A280 ratio to proceed with transfections. What is the best way to clean up this DNA especially as I don't have much.
I would suggest a column extraction. However a simple phenol:ChIsam would do the trick.
Dear Heidi,
Are you sure your DNA is contaminated with proteins? As you said, the ratio 260/280 is rather higher than normal, but when you have protein contamination this ratio should be lower than 1.8, what is accepted as "pure" DNA. There can be however, some other contaminants in your sample (please see the Nanodrop curve for different contaminants in this link: http://www.nanodrop.com/Library/T009-NanoDrop%201000-&-NanoDrop%208000-Nucleic-Acid-Purity-Ratios.pdf )
In any case, as Daniel suggested, your can try purifying again your sample with a column, or I also suggest you to use the SPRI method, which works great as a low concentration DNA cleanup. As an example you could use the AMPure XP paramagnetic beads from Beckman Coulter.
I hope this can help you
Best
Ingrid
Think Zarina meant proteinase K - you would add this at the start and incubate prior to running through kit.
But if you're using the QIAprep miniprep kit, it should give quite clean preps...
There are things you could adjust in your procedure:
- Ensure lyseblue added to make sure your lysis and neutralisation is complete.
- Add less bacteria for plasmid prep
- Do not incubate lysis step for more than 5mins
- Perform the optional wash with PB
- Ensure all ethanol from PE step is removed prior to elution - add an extra spin for 1-2min after decanting first PE flow through. And stand tube in new collection tube for 30s prior to adding EB directly onto silica membrane.
Hope it helps!
John
If you are using QIAprep miniprep kit, it should give quite clean preps.
1. Can you write the detailed procedure you followed?
2. Elution buffer used
3. 260/280 ratio and 260:230 ratio
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology