RNAse is usually included in the initial steps of plasmid purification and should alleviate the RNA contamination issue. As for the plasmid issue, one possibility is that you could be isolating satellite colonies which can grow on the periphery of plasmid-harboring colonies (as these secrete beta-lactamase into the agar that degrades the ampicillin around the colony). For your ampicillin testing, was the test done in liquid media or an agar plate? Do the colonies / liquid cultures grow noticeably slower?

Aside from that, it may be good to try purifying another plasmid that you know has worked in the past, just to be assured that there is no issue with your reagents or procedure for plasmid purification.

Thank you so much for answering my question Aaron P Landry

I have done my testing in both liquid and agar media. But when it was for agar media just after transformation of the cells it took more than 16 hrs to grow colonies . But when I did subculture from any one of the grown colonies it grew well in less time on ampicillin agar plates and liquid media.

I have attached one picture of an agar plate where you can see the colonies (grown after 16 hrs) after transformation of the cells. Does it seem to be satellite colonies only? I have used big colonies only for subculture.

BL21-DE3 is often not advised for plasmid isolation.

see the following link for a comparable search.

https://www.researchgate.net/post/How_to_overcome_nuclease_activity_in_E_coli_BL21_DE3_while_isolating_plasmid

thank you so much Govindkumar Balagannavar

I agree that BL21(DE3) is not optimal for plasmid purification but it should still yield plasmid. The different colony sizes indicate that there may be different bacteria other than E. coli in your competent cell batch, or something else that causes aberrant growth rates from colony to colony. I would try preparing new competent cells from a single colony, in addition to trying a commonly used propagation strain as previously mentioned (DH5 alpha, TOP10, etc.).