Dear RG members,
I have followed the miniPrep protocol for plasmid isolation for two E. coli strains of OD ~ 0.5 (BL21 and DH5a).
The plasmid concentration measurement using NanoDrop showed a concentration of 2746.1 and 5543.0 ng/uL for BL21 and DH5-alpha, respectively. The A260/280 values are 2.03 and 2.07 for BL21 and DH5-alpha, respectively.
I am wondering if the plasmids are contaminated with RNA or the concentrations are acceptable.
Looking forward to your comments and suggestions,
Thanks and regards
Bikash, Osaka University.
Hi Bikash!
Which kit are you using to perform the minipreps? In which volume are you eluting the final DNA? The A260/280 that you are obtaining is more typical of pure RNA; you often get around 1.8 for DNA.
In my lab we use the wizard SV miniprep kit (Promega) for 2mL of DH5a bacteria, and eluting in 100uL we obtain around 100-130 ng/uL but without any contamination, as the A260/280 is around 1.8-1.85. I suggest you to check your protocol and compare it with the one of this kit, because maybe you are purifying all nucleic acids and not just plasmid DNA. Also, maybe you should increase a little bit your elution volume, so the concentration decreases but the potential contaminants that you may have become more diluted!
I hope this will be a little helpful!
Pablo
Dear Bikash,
How much ml of bacterial culture you have taken at the beginning for plasmid isolation?
Hi.
The concentration of plasmid DNA after a miniprep depends on multiple factors as you have been told in previous responses (final DNA volumen, mL of bacterial culture,...).
Check the type of origin of replication of your plasmids to know what to expect in terms of yield.
I also suggest your running a gel to check the puririty o the DNA.
Be careful when you lyse the bacteria, since you could be contaminating your plasmid DNA with genomic DNA.
Good luck
Dear Dr. De Sir,
Thank you very much for your reply.
I have started my plasmid isolation from a culture volume of 5 mL.
From the above responses, I could understand that there is something wrong in the time scale of treatment of RNAse (I had treated for 2 minutes). I think I should increase the time to ~5 minutes.
Thanking you
Sincerely
Bikash
Dear Drs. Pablo and Pedro,
Thank you very much for your valuable comments and suggestions.
The kit that I am using is attached here.
As suggested, I think my plasmid is contaminated with RNA due to small time scale RNAse treatment (< 2 minutes SOL-I) .
I'll perform a gel run to cross check the contamination.
Thanks and regards
Bikash
Hi.
As you say it could be RNA contamination.
If your plasmid has a relaxed origin of replicaon 5 mL of overnight culture may preoduce too much debris and may be clogging the column, spoiling your DNA purification. Try 2-mL.
Good luck
Hi Bikash
A260/280 ratio theoretically should be 1.86 once you get it we can say your plasmid is pure and has no contamination, but your A260/280 ratio tells there is RNA contamination, so one more thing you have to do is to run the gel and see the RNA band. I would recommend to add RNase 1-5 ul (10 mg/ml) again and incubate for 1 more hour in 37C, do phenol-chloroform purification and finally add an appropriate amount of 1xTE.
Regards
Don't expect much in terms of DNA quality and quantity coming from a miniprep and even less coming from BL21 strains. For more efficient results, you should consider sending us your plasmids to prep. We can prep them for only a fraction of the price offer by other companies. You actually save money if we do it for you! By using our own proprietary protocols, we can not only offer the best price to our customers but also obtain better results than you would with a kit in terms of DNA quantity and quality. See for yourself at https://www.biozilla.bio/plasmid-dna-purification
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