I couldn't able to isolate the plasmid and ended up getting only RNA. After repeating it for 2-3 times also I couldn't able to isolate p21 plasmid. I am trying to sort out the reason for this. I welcome all the suggestions from anyone expertise on this. So that i could able to know in which step i made mistakes repeatedly. I am Making all the solutions for isolation mannually. Not using any Kit for isolation.
The plasmid is in E.coli DH5-alpha. Antibiotic used is Ampicillin . Above information is for your reference.
Alkaline lysis of cells should be limited with minimal gentle mixing for 4-5 min. Neutralize with ice-cold buffer immediately and incubate the lysate on ice for 30-60 min. Remove chromosomal plus protein precipitate by high-speed centrifugation. Process supernatant for precipitation/isolation of plasmid.
I am not sure about protocol, you are using. Attached, find a protocol, that works in our hands (can be scaled up, if needed).
Martin
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