I am trying to digest pET28a by Bam HI and Xho I. I am using 1.5 ug DNA and 1ul of enzymes (which ia 20 unit per ul) and incubated at 37*c for 6 hours and in second reaction, incubated for 2 hours.. But when i ran both digest at 1% agarose gel, with un digested vector.. I found that there are 3 bands in undigested vector, but there were no band in both digests.. I used similar concentration of all during loading in agarose gel..
Dear Chankit,
3 bands for the undigested vector can be caused due to following factors,
1. Band type; "Super coiled" (imagine a circle but twisting around and around until it looks like a "rope"). Because it can "slide" through the gaps in the agarose matrix, this is the quickest moving.
2.Band type; "Linear" is the second quickest migrating option. Breaks in the plasmid might cause the DNA to linearize. This (like the supercoiled) can travel through the matrix very well, but unlike the supercoiled, the ends can "snag" or "catch" on the matrix, slowing it down.
3.Band type, "open circle" is the slowest moving. This structure appears to be similar to the plasmid map used to schedule enzyme digestions. It migrates the slowest because it is "broad" and has difficulties traveling across the matrix. Very first band in the gel. Supercoiled and Open circle arrangements are usually occurs in a gel, but the problem with the linear one, it is generated because of the DNAase in the elution buffer. (quality of your prep)
you mentioned no bands in the digested samples, getting a band can be affected by several factors, DNA concentration of the sample, Enzymatic activity (Inhibirory factors), Availabilty of the restriction sites for the BamH1 and XhoI in the Pet28a vector sequence. Here low DNA concentration of the samples may be the reason.
What is the protocol you are following? Why do you have to incubate the digestion mixture for 6 hours? The final buffer concentration (1X) in the reaction mixture and the pH of the buffer are also critical.
Respected Sir, (Lahiru Madushan)
I used 1.5ug DNA for the reaction. I have also done digestion of gene of interest with the same concentration, I got digested bands in the gene. But in vector, I did not find any band. Also, there are both sites present in the vector for restriction digestion.
Respected Maam, Chandra Somasundaram
I have used the final concentration 1x of digestion buffer and I incubated digestion reaction for 2 and 6 hours but did not get any band in both digests. According to lab protocol I incubated for 6 and 2 hours. Digestion buffer i used according to NEB chart.
Respected Sir, Lahiru Madushan
I used 1.5ug DNA for the reaction. I have also done digestion of gene of interest with the same concentration, I got digested bands in the gene. But in vector, I did not find any band. Also, there are both sites present in the vector for restriction digestion.
- Badges
- Science topic
- Similar topics
- Vectorization