I have purified my plasmid from bacterial culture -prepared from different positive colonies in colony PCR- using Iso-probanol precipitation method, and I ran 2500 ng of the purified plasmid on gel, and this was the result (as sown below). I want to know why I got faint band for my plasmid and got alot ( strong band) of short sequences.
te usual Iso-propanol precipitation method, is it possible that RNA give a band on a gel, when I measured the DNA conc. using Nano drop, it was 5000 ng in all samples.
Hi Haitham! How are you?
This seems that bacterial RNA has been isolated along with your target plasmid. Did you use RNase at the beginning to digest the RNA?
If you use a column-based approach (commercial kit), you can get a highly pure plasmid, as they provide efficient RNase that we can incorporate in resuspension buffer.
Anyway, you can get an exact idea based on 260/280 ratio.
Yes Denise, A260/A280 ranges from from 2,15 to 2,17, but how to solve that?
Hi dear Uma, I am fine thanks,
I was using this column method but the Plasmid yeild was too low (25-40 ng/ul) and I want to confirm my plasmid before going to the expression step, so I decided to go for the iso-propanol precipitation method to increase the plasmid yield, any tips regarding to this?
I guess you had used the mini-prep version of a kit. You may try a midi-prep version, which would possibly increase the yield by several folds. Meanwhile, which vector have you used? I wonder the copy number of the plasmid must be too low.
And what is your downstream work? Transform into bacteria (which requires only a few ng of the pure plasmid that is preparable by your available kit) or transfecting mammalian or another cell line (which obviously needs high-concentrated DNA in pure form)?
Still, you have an option, why do not you mix the RNase powder (few mg) into your resuspension buffer that you used before lysing the cells, so that you can digest and get-rid-off all the RNA contaminants!
Good luck.
Yes Uma, I am using the mini prep, this is what is avilable right now in the lab.
The plasmid that I am using is pETM-41, the copy number is Un-known, th down stream work is to transform e-coli(rosetta strain) and upto 38 ng showed no transformation.
I will try to find RNAse and see what happens.
Thanks dear Uma for your kind help.
38 ng of plasmid should be more than enough. You may have to check your steps in transformation.
Check the following:
Precooling
Heat shock [45 s to 1 min at 42 C and immediately on ice for 2 min]
Supply of SOC (this is preferred)
1 hr vigorous shaking at 37 C
Spread on plate with specific antibiotic
You can get a nice efficiency if you consider these steps.
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