I am getting a non specific band in PCR. So, I want to do hot start PCR. I want to know while doing hot start PCR manually, we have to add polymerase after initial denaturation step of 94 degree for 10 mins? or at any other step? and I also want to know tha. It is generally said that when you set PCR reaction at room temp. Then primer binds to non specific site on template. So, I have a doubt that, at room temp. DNA is double stranded, so how can primer bind to the template at this temp?
For "hot start", we simply set up the mix without enzyme and add normal PCR polymerase enzyme of your choice at the start of the first anneal step (after initial melt) so that any polymerase activity cannot have acted on material that annealed at a temperature below your selected annealing temperature
First of all there are many ways to try avoiding your unspecific bands. I think it is not necessary to reapply more enzyme.
You could try a temperature gradient PCR reaction usually starting with 10°C higher than your amplification temperature or try higher MgCl concentrations which is resulting in higher stringency of your system. A touchdown PCR protocol could be helpful too.
The DNA template after denaturation is not a double strand at room temperature, since the buffer contains (NH4)²SO4 which is binding the single DNA strands and hampers the rehybridization.
I totally agree with Stephan Huehn. Instead of directly jumping onto hot start PCR and that also manually, it would be always better to try to decrease the non specificity as he mentioned above.
Dear,
Lalita,
We are not adding any kind of DNA polymerase in between step of PCR.
hot start mean Our Taq DNA polymerase is inactive at room temperature, as soon we reach more than 70 degreeC its become active by relieving blocked active site of polymerase.
primers only anneal with each other in reaction mixture and can give chance to polymerize at room temperature only.
As you told you are suffering from nonspecific amplification in your PCR.
you can use more efficient and reliable poymerases such as
https://www.neb.com/products/m0530-phusion-high-fidelity-dna-polymerase
http://www.thermoscientificbio.com/pcr-enzymes-master-mixes-and-reagents/phusion-high-fidelity-dna-polymerase/
and also play with rising Ta of your primers in your reaction which can help you to amplify your desired amplicon.
all the best
You could try a temperature gradient PCR reaction usually starting with higher than your amplification temperature or increase MgCl concentrations which is resulting in higher stringency of your system. A touchdown PCR protocol could be helpful too.
Dear Lalita,
You have to provide PCR master mix in cool condition (on the Ice Pack), not at room temperature.
For Hot start, you can add Taq enzyme after Initial Denaturation or can add a Amplivax pellet after providing your master and then add Taq and DNA.
But, for getting specific band you can set a touch down or gradient PCR for your work.
All the best
As pointed out in other answers, your nonspecific band could be related to other things than hot start. However, if other nothing else solves the problem and you don't want to use a HotStart polymerase you could first try the cold start mentioned (setup on ice, then directly to denaturation temperature). If you want to use manual hot start the best way is to add mineral oil over the reaction and add the polymerase in a sufficient amount of medium without touching the oil. For instance, if you have a 50 ul reaction you can split the raction components in two: have 25ul in the bottom (template, buffer etc.) undder 20 ul oil, and then pipette the remaining components (25 ul polymerase, buffer etc.) over the oil. By not going through the oil you don't need to change tips between samples. The remaining components will easily go through the oil as long as the volume is at least 15 ul.
Doing it this way it is best to add the polymerase at the beginning of the initial denaturation rather than at the first annealing step since the temperature will momentarily drop a few degrees upon adding. Adding at the annealing step could drop the annealing temperature causing nonspecific primer binding.
In the 90's before HotStart polymerases became available I did manual hot start this way for many years with considerable success.
Hi, Lalita before going for Hot start PCR, have you done gradient PCR using different annealing temps and Mgcl2 concentrations? if not go for it 1st and select the best amlicon taking both parameters in to account. I do not think putting reaction at RT will give more non specificity. Instead of manual hot start PCR I will recommend to go for touch down PCR. Good luck
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