I want to test to see if my cells do indeed have the plasmid with the insert transformed in them, so I plan to confirm this using a PCR. I've been told we can't do a mini-prep to isolate the plasmid from BL21 cells, I'm just wondering what is the reason for that?
You can make plasmid mini-preps from BL21, it is a myth that you can not. I have done it hundreds of times. The problem is that most E. coli strains labs use for cloning purposes carry a mutation in EndA, which is a periplasmic nuclease in E. coli. BL21 does not carry that mutation so expresses the nuclease. So if the nuclease is not removed then your plasmid DNA might be digested. You need to either use a miniprep kit designed to be used with an EndA+ strain or add in some deproteinization step to remove it such as phenol extraction. Another approach is to NOT add an RNAse step to your miniprep procedure, it turns out that RNA is inhibitory to the EndA nuclease and having the RNA present protects your plasmid DNA.
Hi Talia,
BL21 cells are not designed for plasmid purification but protein expression. BL21 lacks in some proteases but has the endonucleases present. You can make mini-preps and try to isolate the plasmid, but there is a risk that your plasmid may be digested. If you decide to do it anyway, make sure that the plasmid is not cut, for example by electrophoresis.
Hi,
If it's only to confirm presence of your construct, you may consider a colony PCR.
You can make plasmid mini-preps from BL21, it is a myth that you can not. I have done it hundreds of times. The problem is that most E. coli strains labs use for cloning purposes carry a mutation in EndA, which is a periplasmic nuclease in E. coli. BL21 does not carry that mutation so expresses the nuclease. So if the nuclease is not removed then your plasmid DNA might be digested. You need to either use a miniprep kit designed to be used with an EndA+ strain or add in some deproteinization step to remove it such as phenol extraction. Another approach is to NOT add an RNAse step to your miniprep procedure, it turns out that RNA is inhibitory to the EndA nuclease and having the RNA present protects your plasmid DNA.
Dear Talia
i'm agree with the answers of Michael and of Johanna.
Moreover some people believe that expand and purify DNA for BL21 is also more risky because those strains are not recA deleted strains and in many cases the protein can be expressed also with out induction therefore it is possible that the plasmid stability in this bacteria is low and ta mutation or recombination has an higher probability to happen in this strain. However i also extracted several times plasmid from BL21 and it worked well.
Ciao
Manuele
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