4 replies to this topic

[ocean_sky83]

Hi all

I am expecting my pcr product size to be 550bp but I obtained something almost 600bp. I have a positive control that works well and shows the expected size. Could this be due to some kind of insertions in the region?

I would think that the 600bp was unspecific however it was amplified very brightly as a single band. For that reaction I also added pcr enhancing agent to it. Before that, it was showing a very faint band, almost can't see it. I know that pcr enhancing agent such as betaine is used when you have a dna template with high GC however my template didn't have high GC but I used it as i wanted to know if it will make a difference.

I was just wondering why the amplification get a single bright and clear band when it is unspecific? (since it is not the expected size)Isn't pcr enhancing agent supposed to increase specificity and wouldn't allow amplification of non specific band??!?

Thanks!
Have a nice day

[jessh]

It might be the band you want?? I would imagine that additives like betaine and DMSO would affect the mobility of DNA through a gel by retarding it and making it thus making it appear bigger. I am not sure if the weight is enough to equate to a 50 bp difference but you could try cleaning up your product and running it again to see if it shifts down. I would just gel extract the band and cut it with a restriction enzyme. This way you can check if it is your desired product or something else! - quick and easy and you will get an answer.

Also, even if your template doesn't have high GC it still may have regions of complementary that could cause secondary structure, and in this case the additives will help.

Hope this helps!
jess

[Adrian K]

just to play safe, is it possible for you just clone the 600bp fragment and sequence it?

[HomeBrew]

It might be better at this stage to just sequence the PCR product directly rather than cloning it first, just to see if it's what you want before going through all the effort of cloning it.

[Adrian K]

Agree with HomeBrew...
my mind must be stuffed with food and thought of something else...LOL