I have done a ligation reaction and transformed DH5alpha cells using the ligation mixture.
I got colonies in the amp+ plates and so inoculated those in the liquid broth containing ampicillin (of same concentration). I did a miniprep for the plasmid isolation and the Nanodrop readings I got after that was also looking OK, as in the A260/280 ratio was around 1.9 and A260/230 ratio around 2.
I wanted to cross-check the concentration of the plasmid, and ran it in agarose gel. I did not see any band instead I saw a blob.
Is it a possibility that the DNA is getting degraded? I have eluted in Nuclease free water (preheated to 50 C).
Can any of you help me out?
Can you show us your gel ? How much ng/ul did you get from the nanodrop reading ?
If you had good nanodrop ratios, it is unlikely your DNA would degrade just like that. If it had, you would see a "smear" on your gel, not a "blob". I always elute in water and my DNA stays good for over a year.
Try digesting that DNA with enzymes you know will cut your plasmid in 2-3 sites (in the backbone and in the insert) to confirm its identity.
I concur that it would be helpful to see a gel picture in order to better diagnose. It may also be that you have vastly overloaded the gel so that it looks like a blob. If you are running uncut or singly digested plasmid you only need 100-200 ng of DNA for a nice bright band.
Thanks for your replies.
The DNA in the agarose gel appeared like a smear.
The following is the picture. I have also digested with an enzyme that cuts only once in the insert.
The nanodrop readings range from 122ng/ul to 350ng/ul. So, according to this I would have loaded ~600ng in each well which does not seem from the gel image.
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