6 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

[leelee]

Can you post up your gel? You may have the correct product, but your gel isn't good enough to properly resolve a 50bp difference? If your band is quite bright and thick, this could also complicate your size estimation.

[ocean_sky83]

leelee, on 11 October 2010 - 12:38 AM, said:

Can you post up your gel? You may have the correct product, but your gel isn't good enough to properly resolve a 50bp difference? If your band is quite bright and thick, this could also complicate your size estimation.

Sorry! I do not have a gel image now.. But i am very sure I didn't estimae the band size wrongly or due to the gel quality didn't resolve the 50bp difference.

I used to experience pcr product slightly larger (~50bp) than my expected product size ( but faint band )... so what can be the case? My question actually is whether using pcr enhancing agent when it is not necessary as there was not high GC in my template so what kind of effect can it do to my result? As i have read, it removes secondary structures so that your taq can bind better so to improve the yield. But the size wasn't the expected size. The difference is about 50bp, however using that it makes a single bright band amplification at approximately 50bp bigger than what I am expecting..

Is it like anything to do with binding to the wrong site? or perhaps like insertions in my target, or like some gene variant that kinda stuff?

Thanks!
Have a great day

[ram]

I agree with leelee. It can be the mis-interpretation of the siez. I remember, once I had similar problem, then I loaded different quantities of the same PCR product on the gel. And I got the picture which looked something like this. You can try the same

[ocean_sky83]

ram, on 12 October 2010 - 02:33 AM, said:

I agree with leelee. It can be the mis-interpretation of the siez. I remember, once I had similar problem, then I loaded different quantities of the same PCR product on the gel. And I got the picture which looked something like this. You can try the same

What if i have used the same amount 5ul pcr product for both my sample and positive control but the band looked of slightly different size. You mean if I load in more pcr product they may look of differently?

But why?

[ram]

ocean_sky83, on 12 October 2010 - 05:36 PM, said:

ram, on 12 October 2010 - 02:33 AM, said:

I agree with leelee. It can be the mis-interpretation of the siez. I remember, once I had similar problem, then I loaded different quantities of the same PCR product on the gel. And I got the picture which looked something like this. You can try the same

What if i have used the same amount 5ul pcr product for both my sample and positive control but the band looked of slightly different size. You mean if I load in more pcr product they may look of differently?

But why?

Its not about volume of your PCR product, rather its about the quantity of product that you get. you shud load such volumes of your positive control and test samples that intensity of the bands should be similar for test and positive control. In other words, comparing sizes of bands having different intensities can give misleading results

[ocean_sky83]

ram, on 13 October 2010 - 12:27 AM, said:

ocean_sky83, on 12 October 2010 - 05:36 PM, said:

ram, on 12 October 2010 - 02:33 AM, said:

I agree with leelee. It can be the mis-interpretation of the siez. I remember, once I had similar problem, then I loaded different quantities of the same PCR product on the gel. And I got the picture which looked something like this. You can try the same

What if i have used the same amount 5ul pcr product for both my sample and positive control but the band looked of slightly different size. You mean if I load in more pcr product they may look of differently?

But why?

Its not about volume of your PCR product, rather its about the quantity of product that you get. you shud load such volumes of your positive control and test samples that intensity of the bands should be similar for test and positive control. In other words, comparing sizes of bands having different intensities can give misleading results

Thank you ram, got it!