I digested my lenti guide puro plasmid. The only modification in this plasmid is that it has blasticidin resistance gene instead of puromycin. I get the desired bands at ~8kb but I do not see the 2 kb band. However, I get a 1.2 kb band and 0.8 kb band. I used FD EsP3I from thermo fischer and used 3ul of Esp3I for 5ug of plasmid along with 3 ul of FAST AP, 6 ul of FD buffer and 0.6 ul of 100mM DTT in a 60ul reaction and digested it overnight (approximately 16 hours). When I gel isolate my 8kb fragment (lanes 2 and 3 in the image) and transform it into Stellar cells, I get a lot of colonies on the plate. In fact, the plate is full of colonies. Also, I tried it with two separate midi preps of the plasmid and I get smearing on digesting for one of the preps (lanes 5 and 6).
In the figure, lane 1 is undigested lenti guide blasticidin plasmid. lane 2 and 3 are digested lentiguide blasticidin plasmid with Esp3I from midi prep1. Lane 4 is 1 kb plus DNA ladder. Lane 5 and 6 is smeared lenti guide blasticidin from midi prep 2. Please help!
Maybe you have a restriction site, of one of your restriction enzymes, inside the 2kb fragment, and that why you get 1.2 and 0.8 fragments. In such a case you should use another restriction enzyme. check before that you dont have sites of ths enzyme in the desired fragment.
Your gel looks very unusual and I suspect you might have a mixture of plasmids. First of all, the uncut lane is very suspicious. It is true that supercoiled plasmid runs faster than open circle or linear plasmid but not like what you are seeing. If the plasmid is 8kb in size then maybe the supercoiled would have an apparent mobility of 4 or 5 kb but not 1kb.
Why don't you take some of each midi prep and do some diagnostic digests, uncut, cut with Esp, and cut with some enzyme that should only digest one time to linearize and see if it makes sense or not.
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