4 replies to this topic

[fortunate]

Hi, we bought a DNA extraction kit. The extension step in its PCR protocol (for multiple amplicons amplication) uses 'Extension: ramping from 55 'C to 72 'C for 5 min'.  Can someone tell me what this means? I did a gradient temperature, but it seems not the case. Thanks!

[swanny]

From what you've said, I presume you are working with different primers in the same sample, the same primers in a few different samples (with varying degrees of homology), and/or you are trying to get different regions of the same template DNA amplified using common primers, or something else like that, right?

Not knowing which machine you are using, I can't start to give great detail about how to program this ramped extension. What you could do is to have 4 or 5 extension steps in each cycle. Each step would last for 60 sec, say, then increase by a degree or two. The range for  the extension step is pretty big, going from 55 (which will give almost no polymerase activity) to 72.

Can you give us any more details of the project?

[fortunate]

Swanny, Thank you very much! Hope to see you again here.

swanny, on Sep 1 2009, 11:34 PM, said:

From what you've said, I presume you are working with different primers in the same sample, the same primers in a few different samples (with varying degrees of homology), and/or you are trying to get different regions of the same template DNA amplified using common primers, or something else like that, right?
Thank you so much for your help! I was doing knockout genotyping using mouse tail DNA. Yes, I used two pairs of primers, one for wild type, one for knockout.
Not knowing which machine you are using, I can't start to give great detail about how to program this ramped extension.
I am using MJ Mini personal thermal cycler from Bio-Rad. What you could do is to have 4 or 5 extension steps in each cycle. Each step would last for 60 sec, say, then increase by a degree or two. The range for  the extension step is pretty big, going from 55 (which will give almost no polymerase activity) to 72. Could you explain a bit more what is the purpose to do this kind of extension? What will be the difference if I just use a fixed temp like 72 'C?
Can you give us any more details of the project?
I was using a kit from GeneScript http://www.genscript...out_Enzyme.html. They mix the buffer with their polymerase. It is the kit's recommedation that I use that Extension temperature range and 5min. My first PCR trial took over 7 hours!

[Adrian K]

Hi fortunate;
I had a look through into the protocol, this is my understanding regarding the protocol

Let's assume that your thermal cycler had a ramping rate 1C per seconds, and your annealing temperature is 55C

heat From 55C increase to 72C had taken 17seconds. so for the extension time you key in 283 seconds to complete 300 seconds (5 minutes)

or you had a ramping rate of 0.1C per seconds,
heat from 55C increase to 72C had took 170 seconds. so for the extension time you key in 230 seconds to complete 300 seconds (5 minutes)

This is what I understand.

Also, since there are some amplification going on at temperature before 72C, i guess the manufacturer want to make sure the amplification does not exceed 5kb range (Assume their taq activity is 1kb / minute) to avoid over used of their taq.

Just my 2 cents. Correct me if I'm wrong.

[swanny]

fortunate, on Sep 4 2009, 02:08 AM, said:

Swanny, Thank you very much! Hope to see you again here.

swanny, on Sep 1 2009, 11:34 PM, said:

From what you've said, I presume you are working with different primers in the same sample, the same primers in a few different samples (with varying degrees of homology), and/or you are trying to get different regions of the same template DNA amplified using common primers, or something else like that, right?
Thank you so much for your help! I was doing knockout genotyping using mouse tail DNA. Yes, I used two pairs of primers, one for wild type, one for knockout.
Not knowing which machine you are using, I can't start to give great detail about how to program this ramped extension.
I am using MJ Mini personal thermal cycler from Bio-Rad. What you could do is to have 4 or 5 extension steps in each cycle. Each step would last for 60 sec, say, then increase by a degree or two. The range for  the extension step is pretty big, going from 55 (which will give almost no polymerase activity) to 72. Could you explain a bit more what is the purpose to do this kind of extension? What will be the difference if I just use a fixed temp like 72 'C?
Can you give us any more details of the project?
I was using a kit from GeneScript http://www.genscript...out_Enzyme.html. They mix the buffer with their polymerase. It is the kit's recommedation that I use that Extension temperature range and 5min. My first PCR trial took over 7 hours!

As far as I can tell from the protocol, the 55-72 ramp is for a multiplex assay using their primer sets. In your case, where there are only 2 sets of primers to consider, you could use a narrower range, if you like. I think you need to be able to make the temperature change, rather than just have a single temp.

As for  the 5 minute extension, my first guess would that the enzyme is not very fast at the lower temperatures (anything below 65, as memory serves), so for the first couple of minutes your DNA polymerase is crawling along the template. Then as the temperature rises above 65, the reaction rate increases. If your primers have similar Tms, you might be able to shorten the extension time, and also reduce the temperature range.

What extension times and temp do you use for single primer sets? You should use the longer extension time, and lower temperature as your first parameters.

Edited by swanny, 07 September 2009 - 10:48 PM.