The possibility of the pellet in the left tube being an antibiotic-resistant contaminant should be seriously considered, unless there is a specific reason to believe that the cloned gene encodes some kind of pigment. The first thing to do would be to prepare a new stock of competent cells starting from a validated stock of the recipient strain and to perform the transformation once again (you could use the same ligation mixture, if some leftover is still available).

Your color is likely due to tRNA presence (you can see it at the bottom of your gel), which is sometimes yellowish or pinkish when isolated from a native source. tRNA is a common contaminant in alkaline lysis, as is escapes being precipitated with denatured proteins the same way plasmid DNA does.

However, this should not have an impact on your cloning procedure.

There is something very strange about your gel, genomic DNA usually presents as a smear and not as discreet bands. And your vector is running with the same mobility as the bands near the top of the gel. Seeing some tRNA at the bottom is pretty typical. What percent agarose gel are you running? My guess is that you do have some plasmid bands at the top but you are not resolving them with your gel. Also what size is your vector and insert?

Michael J. Benedik

thank you very much for the response. I am doing a 2% Gel. I also just loaded all the oalsmid that i had (30uL) and got a faint band at the expected size. So could it be that the concentration was just extremely low for some reason?

A 2% gel is really only suited for looking at small fragments. That is why the top of the gel is so compressed. I think if you ran a 0.7 or 1% gel you would see bands much more clearly. I'm not sure what size your insert is though, but if it is more than 300 nt you should be able to see it fine on a lower percent gel.

Admittedly this is not going to fix the low DNA concentration problem, that is a different issue.