Recently, I tried to cut a 700 bp DNA to 500 and 200 base pairs by ECOR1 restriction enzyme. After that, I electrophoresed my sample through a 1.5% agarose gel and the obtained result was as follows:
1. Regular bands such as 200, 500, and 700 bp.
2. Surprisingly, a sharp band with 1400 base pair (double-checked)
I can't figure out how it is possible. There wasn't any additional material such as ligases but restriction enzyme.
Hi, then cut out the band and sequence it. After blasting the sequence you will know the result.
Cheers, Nadine
Hi, then cut out the band and sequence it. After blasting the sequence you will know the result.
Cheers, Nadine
Hi,
First, be sure if your enzyme label or contet is correct. You can also cut your DNA with other restriction enzymes to see correct digestion.
Good luck
Haydar
Hi, it could be worthy to check your initial material (before digestion) on a gel and see you if you already see this 1400bp band. The other thing is as Nadine has suggested, Just cut out your bands and sequence them.
Good Luck,
Ameera
I wonder how will you sequence the 1400bp fragment, if you dont know what it could be..since you cant amplify it with any primers ...i would suggest rather to check you enzyme..whether its contaminated...mix equal amount of enzyme (that you used for digestion)..dilute with water,,add loading dye..n run it next to your digested sample..may help...
Is there a possibility that your undigested molecule is a piece of linear DNA with cohesive, and rather longish self-annealing overhangs? If so, undigested molecules might be annealing during the restriction digest, as often happens with lambda DNA fragments bearing cos ends. This should be easy to rule out by pre-heating the reaction before loading it on the gel (e.g. 10 min. at 65*C).
Another possibility is contamination of one of the components of your reaction with foreign DNA. Set up a restriction digest without DNA and check it by electrophoresis to see if anything shows up.
Hello Munsouri,
you may need to cut off the 1400 bp band from the gel and re-digest it with your Restriction enzyme. Then, run a gel. If you will get 700 bp and 1400 bp band, the 1400 came from overlapping of 700 one.
Good luck
You may necessitate incising the 1400 bp band from the gel and re-digesting it with your Restriction enzyme. Just you may have done once repeatedly the similar experiment. Then, run a gel. If you will get a 700 bp and 1400 bp band, the 1400 came from partly covered of 700 one. Supposed be undigested molecules might be improve or alternate the little bit cooling condition during the restriction process, This should be easy to regulate out by pre-heating period (15 minutes) as well as temperature(s) (30 to 60°C) the reaction before loading it on the gel.
Dears, although Ardalan got the answer to his "strange" band of 1400 bp, I want to point out that sequencing without amplification of the target (aka amplicon sequencing) is perfectly possible (de novo). But I am writing here for a different reason. Do you really believe it's helpful to recommend such an expensive technique just for checking some kind of artifact? Common. I voted one of the contributors in whom suggestion I sensed a genuine desire to help and not writing for the seek of having one more post.
@Arthur: it is a real one. I can also show you a fake gel electrophoresis or even a different process manner. So if you think it is untruth, I can't change your mind.
- Badges
- Science method