I am a research student trying to harvest DNA from a 2400bp plasmid with a ~750bp insert. The plasmid backbone is noted as high copy. The sample is from AddGene.
I prepared LB with Kanamaycin in it - it is fresh and I autoclaved before adding the antibiotic to ensure sterility.
I grew DH5Alpha E. coli with plasmid in LB, streaked that on a plate (also with Kanamycin), and then grew cultures from a single colony, and finally made a large overnight culture.
There is at least 50/50 cell volume to air, so I do not think aeration is an issue. Also, liquid cultures are fresh - never grown for more than 24 hours.
I have tried completing plasmid prep kits from Zymo (I have tried Zyppy mini and ZymoPure midi), to absolutely no avail.
I follow the instructions meticulously, including temperature. I have tried heating buffers to increase DNA yield. I pellet cells, resuspend in water, lyse (but not too long!), neutralize, pellet, wash the supernatant, and elute with elution buffer provided in the kit (I have not tried to elute with water).
UV spec DNA concentration ratios are terrible. Just to make sure, I have also checked the eluted DNA sample in both 2% and 1.5% agarose gel stained with EtBr. Still...nothing. Only the ladder shows up
Help, please! What is going wrong? I know that there is DNA in the bacteria...but where is it going?