Hello everyone! I have been trying to do a Maxiprep on my e.coli but I was unable to get any plasmids and the reason is that my e.coli are not being lysed whatever I do ( ex: increase Lysis buffer incubation period to 5 min, 10 min, and 30 min). I also used new kit with new lysis buffer but the result is the same! I am using XL-1 Blue e.coli and I got really good results from that before but now it is not. I also tried shaking more and even used smaller culture volume but now difference. the lysis buffer looks good and everything I do is to the book. what do you suggest?
This is the photo of my pellet after I added all the buffers ( resuspension, lysis, and binding buffers) and then centrifuged appropriately. ( as you can see there is the e.coli pellet not being lysed)
I use this when I need to make new or fresh reagents...
https://people.mbi.ucla.edu/sumchan/qiagenbuffer.html
You can try that...I've used really old kits and the reagents worked fine. I hope you figure this out, try making your own lysis reagent from scratch.
After lysing the solution becomes extremely viscous, like snot, correct?
I agree with Sebastian Schmitt
that the first thing I would try is to make and use fresh lysozyme and give it a few minutes to work before adding the lysis buffer. You can enhance lysozyme action by letting it work at warm temperatures instead of on ice (room temp to 37C).- Badges
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