Hello, I have propagated a plasmid form a single colony (size around 15 kb) using small scale culture (2 ml of TB with selected Antibiotic) and purified it using Mini-prep kits ( FastGene Plasmid Mini kit) and verified it using Restriction Enzymes ( HpaI and MluI ), It gave a correct cutting pattern as seen in the Gel electrophoresis picture. However, when I propagate it using large scale culture (100 ml of TB with Selected Antibiotic) and extract the plasmid using Midi-prep Kits (Macherey Nagel), I could not obtain the correct cutting pattern, instead I only see a smear (the 2nd picture).
I tried several times to change the culture conditions ( e.g increasing the A.B conc in the growth medium, or decreasing the shaking speed, or optimizing the incubation hrs. (12~16 hr). However, I also obtain the same result.
Any tips to overcome this problem and obtain the correct plasmid would be much appreciated.
Hi Rofaida,
I am wondering if this problem has been solved for you?
I am having eactly the same issue like you that's how I found your question. I noticed this issue because I kept failing the sequencing result for the midi-prep sample. @I am still troubleshooting this issue, but I think it is highly possible that what you saw in the gel of your maxiprep is actually gDNA, which somehow got liberated from the precipitation after neutralization. I am using exactly the same midiprep kit from Macherey-Nagel, and from what I read in their manual, they really stressed that no shaking or vortex during the neutralization step to avoid gDNA release and also to not damage the plasmid DNA since it is very vulnerable. and I did shake very hard during loading the sample... I think I will try to not shake but only inverted a few times to see if that can solve this problem Rofaida Soliman
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