E-coli strain and plasmid incompatibility...? - is there something like that? (Aug/29/2006 )
Kathy, is there a reason you are cloning in a BL21 strain?
I have typically found better yield of plasmid from the usual cloning strains (JM series, DH5a, etc). once you have confirmed your clone via miniprep and sequence, it is then a simple matter to transform it into the expression strain when you want to make the protein. but just for cloning, use a strain designed for cloning. when your plasmid is purified, you can transform it into any old strain you like for further usage
thanx a lot everyone,
aimikins, actually i have no reason other that i have used BL21 stain for cloning before and it worked perfect.... ...and DH5alpha that they have here in the lab have been producing weird results lately. yesteday freind of mine here said they have JM109...well i knew that al last, better late than never ...so im electroporating into them now....let's see what happens.
phage im sorry i got confused with these markers....actually here too origami strain doesnt have endA-....so i guess it means that they are endA+....
thank you spanishflower, i printed it out..
I don't have it with me, so I can't quote page numbers, but check out the NEB catalogue. You want a strain that is Rec-, but also EcoK or EcoB R-. These gene groups will not restrict foreign DNA (the R part), but may methylate your DNA to E.coli patterns (EcoK or B M codes for a methylase). In addition, look for cells that are Mcr negative. I lost the first 6 months of my Masters before my PI and I realised the cells we were using were chewing up the DNA.
As a few others have said, your best bet is to go through something like DH5a, purify the plasmid and transform into BL21 for protein expression purposes.