We have transformed the test plasmid of pET-32 Xa/LIC cloning kit with E. coli DH5 alpha competent cells. After 24hours of incubation on LB medium (50 µg/ml ampicillin containing medium) at 37°C, bacterial colony was found. We isolate the test plasmid from the colony by plasmid purification kit and check DNA bands on agarose gel (1%). But we don't know the size of the test plasmid, that's why we are confused whether the transform of test plasmid in E. coli DH5 alpha competent cells was successfully done or DNA bands are because of genomic DNA? (Result of agarose gel is attached)
Any comments or suggestions are welcome..!
It is not clear what the size markers are but this looks like a supercoiled plasmid. No I do not think you have the genomic DNA here. Try cutting with a restriction enzyme and see what you get. Your plasmid should be about 5926bp. You can get a map online just by googling the plasmid name. The supercoiled plasmid does not run at the at the size of the linear plasmid, it usually runs at a lower MW.
Hi, here the map https://www.snapgene.com/resources/plasmid-files/?set=pet_and_duet_vectors_(novagen)&plasmid=pET-32_Xa_LIC. And I do agree with the answer above, you cannot determine a size of plasmid without cutting it. Pick an enzyme from the MCS, generally they are unique site on the vector. Here you can use EcoRI, EcorV or BamHI which are easy enzyme and pretty cheap.
Thank you sir for your valuable reply.
As per gel result (image attached previously) right now bands are coming between 2000-2500 base pairs.
For your reference as per cloning kit manual, size of the Xa/LIC vector is 5926bp. There is not mentioned any name or size of the test plasmid given in the kit.
Is there any other way to check if it test plasmid or not?
It is difficult to judge about size on your picture as we don't know which ladder you used. Is it the 1kb ladder on the left ? And you have to cut the plasmid to determine the size. Also to be sure that you have the good plasmid you can do a restriction map. You decide of pair of enzymes which will give you a "pattern" of different sizes. You can also send it to a compagny to sequence a part of it but it takes a bit more time.
- Badges
- Science topic