Hi all,
I have been trying to do cloning recently. After lots of effort, I finally come to do the mini prep with iNTRON kit. When we shake the colony in 5 ml of LB with 5ul of ampicillin. There was bacterial in the tube and we could also measured the concentration (~300ng/ul) of the plasmid with normal A260/280= 2.13 and normal 260/230. However, when we load 1ul or 2ul of the plasmid to run a 1% agarose gel, no bands were seen. How could this happen?
Thank you so much!
Hi,
300 ng/ul seem normal for isolate plasmid using an extraction kit.
How many times you did electrophoresis? And you see the band of DNA ladder? If not, maybe you forgot adding dye for electrophoresis ^^
Hi Le Minh Thong
Thank you so much for your help! We repeated the results and increased the plasmid from 1ul to 5ul. However, as you can see in the photo, there are 2 bands. Presumably, the top one is the plasmid, despite very weak. But we have no idea what are the bottoms bands. Do you have any ideas?
Thanks!
Hi!
The bottom band is RNA from bacteria and this RNA may also affect the measurement of nanodrop. You can remove it with longer incubation time of resuspend buffer (with RNase) or increase RNase amount in the buffer. :D
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