you're doing a mini prep from BL21? This strain is rather for protein expression. Why don't you try something more suitable for cloning/plasmid propagation like DH5alpha?

Thanks marcin! just out of curiosity, could the bands from the gel be produced because i used an unsuitable cell line?

don't think the problem because of the cells you are using, because even when you you use BL2 you make mini prep to be sure that U have transformed the cells before U express the protein. Did you check if the enzymes you use are the buffers are suitable for double digestion?

.

Hi Rana, I've already checked for multiple digestion sites on NEB cutter and there are no other additional sites on both my plasmid and insert. I've also checked for star activity and there are none. besides that, the buffers I'm using are also suitable for both enzymes.. but thanks for the suggestion!

so you're trying to cut out the insert from the plasmid, am I right? or the gel you're showing is the empty plasmid that you are trying to linearize in order to ligate your insert into it?

Hey Liyana, since you are using BL21, a strain expressing endA, an endonuclease which will damage your miniprepped plasmid DNA, my question would be: When you did your miniprep with the QIAGEN Miniprep kit did you do the PB Buffer step? You have to do this with BL21. Also you have to work fast after the lysis step with the P2 buffer because of the endA activtiy. endA activity may explain the strange pattern of you DNA gel after digestion.

Liyana,

A complete digestion will look as a series of bands whose fluorescence decreases monotonically from higher to lower molecular weight (a digestion produces the same molar amount of each band, but the amount of EtBr bound by each is proportional to mass). Your gel shows either undigested or at best, partially digested DNA; and the three lanes at the far right look like chromosomal DNA. I suspect your preps are not clean enough.

Undigested plasmids preps from recA+ strains (such as BL21(DE3) not only show the usual supercoiled, nicked and sometimes, linear forms, but plasmid multimers (and their three forms as well). The banding patterns in such cases can be quite complex, as you would expect.

BL21(DE3) is a poor choice for cloning work. Better try a recA endA strain; it will save you time and frustration.

Hope it helps

Hi! Liyana,

As Marcin suggested I would say go ahead with DH5alpha which is best for restriction digestions, ligations and cloning work. In our lab we too use BL21 only in protein expression studies. I am using DH5alpha for the past 10 years for all my regular clonings, never had any problems. It should be a smooth ride with it i guess.

Good Luck,

Madhavi

You should get good results using

500 ng your plasmid in 30 ul volume with overnight digestion.

Hi Marcin, yes you're right. I'm trying to cut my plasmids and see if I have my inserts.

Hi Dong-Jiunn Truong, thanks for the suggestion! I spoke with my supervisor and he thinks the same. I'll try using Dh5 and update with my results. And thanks everyone for the help!

hi everyone, I tried using DH5 alpha and got this. the above picture is a picture initially and the second one was after 20 minutes (because I wanted to size them). I was hoping to get two clear bands (my plasmid-4.8kbp and my insert 568 and 1373 bp respectively) I transformed my ligated plasmid and insert into Dh5 alpha and minipreped them using QIAGEN kit. Then I digested them with the enzymes I used initially to split open the plasmid. And then I analyzed it on a gel.

What bothers me was that, I got 3 bands at first, then run it on a gel, then got 4 bands later!! I asked the people around my lab and no one could explain this. Please help :(

First make sure that your plasmid is pure by just running that without any restriction digestion. Then you try your RE one by one to see whether they are working alright. If not change the source of the enzyme.

Liyana, hope you have resolved this problem, but you seems to have stumbled upon a solution to a problem we face working with bacillus systems.

Shuttle vectors that can be transformed between E coli and Bacillus has very low transformation efficiency transforming to bacillus when generated in E coli recA- strains.

The reason many scientist suggest is, the plasmid being in monomeric form. Bacillus show better transformation efficiency for plasmid preps where the plasmid is in multimeric form.

Since you used BL21 (DE3) a recA+ strain you might have generated multimeric plasmid.

Do you have the weights of the DNA ladder you used in gel-1? Do the higher molecular weight bands corresponds to multiples of the original plasmid?

Let us know this might be a valuable observation.

Hi Liyana

There are few possibilities that come to my mind:

1) When you run your DNA digest on gel, do you take it straight from reaction mix? It is possible that some restriction enzymes remain bound to your DNA which affects the mobility of your DNA and you see bands larger than expected. To check this add SDS to your DNA (the gel loading mix) to final concentration of 0.1% to 0.5% and then run your sample on gel.

2) I always (no matter what plasmid extraction kit I use) ethanol precipitate my plasmid mini preps before downstream work. Your plasmid mini preps usually contain high salt concentrations which may affect your restriction enzyme activity.