manual vs. automatic HotStart - (Sep/01/2008 )
Hi everybody!
Can someone please explain me the difference between "manual" and "automatic" hotstart Taq polymerase?
My colleague here claims that it depends on when you put enzyme and reaction in the PCR machine: manual hotstart means that you keep everything in ice untill the machine has already reached start temperature, while automatic one consists in putting everything in the machine at room temperature and THEN allowing it to reach activation temperature.
Well, I'm not sure that it's the real difference, but I couldn't find any help googling.
I hope I was clear enough...
Thanks in advance for your help!!!
ILA
Mmm, will it be right to call this 'hot start'?
Silly question,
Is it possible to manual hotstart with any taq pol? Or only specific types? ie: with NEB thermopol + DNA taq pol?
vairus Aug 20 2005, 10:36 AM Post #8
I beleive that you can manually hotstart with any enzyme. Just hold everything on ice, start your PCR-program and put in your tubes when temperature of the machine is high enough.
As for MgCl2 of MgSO4. I once tried to do a PCR with an enzyme that normally has the SO4 and changed it for Cl2, and I had a drastically reduced yield, specificity and all that were okay. So depending on your needs (that is if yield is important or not) you can interchange them I think.
Excerpt from this thread http://www.protocol-online.org/forums/inde...manual+hotstart
At least my colleague agrees with vairus...
What I understand by 'manual hot start' is putting everything together except polymerase in the tube. . putting them in the hot block till the temperature if high and then putting in the polymerase.
I think I am seriously wrong there. 
Mmmm, jst another attempt to see if I have understood it right. . senior's help NEEDED though.
Hot-start PCR: This is a technique that reduces non-specific amplification during the initial set up stages of the PCR. The technique may be performed manually by heating the reaction components to the melting temperature (e.g., 95˚C) before adding the polymerase.[21] Specialized enzyme systems have been developed that inhibit the polymerase's activity at ambient temperature, either by the binding of an antibody[9] or by the presence of covalently bound inhibitors that only dissociate after a high-temperature activation step. Hot-start/cold-finish PCR is achieved with new hybrid polymerases that are inactive at ambient temperature and are instantly activated at elongation temperature.
Manual hot-start what I had understood was right. .
Automatic Hot-start is what is underlined. I have been using Taq and that is what it does. . .
Thank you for your help, BB!
I know what Taq and HotStart are (anybody who attends a molecular lab should!).
The matter is: what does it mean "automatic/manual" referring to HotStart?
I found this definition somewhere, but I couldn't understand what this two terms mean...
I know what Taq and HotStart are (anybody who attends a molecular lab should!).
The matter is: what does it mean "automatic/manual" referring to HotStart?
I found this definition somewhere, but I couldn't understand what this two terms mean...
Automatic hot start is when you use a special Taq polymerase that only becomes active when it reaches a certain temperature. For example, I use Promega's hot start polymerase. I can set up my reactions at room temperature, and then put them in the PCR machine. The polymerase doesn't get released into the reaction until it's been incubated at 95°C for at least two minutes. There are different ways companies achieve this: the polymerase can be inactivated by an antibody that is denatured once a critical temperature has been reached, or it can be contained within wax beads that only melt once the temperature is high enough. It is considered 'automatic' because the Taq is automatically released into the reaction once certain conditions are met.
Manual hot start means you mix together everything but the polymerase in your reaction tubes and put them in the thermal cycler. You then manually add the Taq to each tube once the reactions are hot enough. This method is effective, but more time-consuming and annoying, especially if you have lots of samples and need to add Taq to each one.
Does this make send to you?
Ginger
oh! I see...
Thanks for your explanation, Ginger!!!
am attending cell biology lab 
...in fact you already knew it! 