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PCR conditions unknown?


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#1 meedomada

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Posted 27 April 2015 - 12:40 PM

I have the primer sequence from a paper but they did not  mention the PCR conditions, and even the reference they mentioned is not using the same sequence

Could any one help with a method to determine the suitable conditions for the given primers and gene

 



#2 bob1

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Posted 27 April 2015 - 03:59 PM

THis is one of the many issues with regard to science publications and repeatability... often the methods sections are seen as an easy place where you can leave out information - however this is the one section where such information is crucial to repeatability of the experiments.

 

However, for PCR, repeatability relies on having the same conditions, including the same type of PCR machine. You should be able to determine the reaction conditions empirically as you would for any other set of primers.



#3 meedomada

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Posted 29 April 2015 - 01:32 PM

How could I determine the conditions empirically? 



#4 bob1

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Posted 29 April 2015 - 01:40 PM

Work out the theoretical conditions (Tm, salt etc.), then test them...



#5 meedomada

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Posted 29 April 2015 - 01:49 PM

I am sorry for that, but is there a web site where I could find a way to calculate it

I know about the Tm calculations, but not much about other things?

Many thanks for your help



#6 phage434

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Posted 29 April 2015 - 02:50 PM

1. Decide what enzyme to use. Below, I will assume Phusion.  Details will be different for other enzymes.

2. Highly recommend you use a master mix, which avoids many problems.

3. High GC organism? If > 60% GC content, use a high GC master mix, otherwise, a normal mix

4. How long is your amplicon?

5. Your cycles will be:

  98 for 30s

 then 34 cycles of:

    98 for 10 s

    55 for 15s

    72 for 30s per Kb of your amplicon length

 

Finally, 72 for 3 m

 

This will probably work.  If not, then adjust the temperature of the 55 degree stage. Lower if you have no bands, higher if you have too many bands or a smear.

Common errors include using too much template DNA.  Less is more.



#7 Trof

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Posted 30 April 2015 - 11:50 PM

I would probably disagree about the necessity to include PCR conditions these days. It is historically true, that all the primers, the PCR mix and machine (programe) have some kind of influence over success of PCR. So to be absolutely exact, you would need to specify all of this.

But then what, using different polymerase/mix if it's not just some general Taq (and the authors are not using general Taq possibly anyway) and having a different cycler (I have seen complete protocols listed, almost never the cycler in classic PCR) will cause you to change the conditions, since you cannot simply duplicate them.

This is still something you need to do and chceck (specifity etc.) on your own.

 

However now, with PCR mixex that are able to work accross range of temperature, that are optimized for specificity, these detailed conditions lose point. I got into this problem, when we were supposed to make a method section to a book, using primes from different labs, I was putting them together for other labs that wan't to sequence those genes. But I wondered how meaningful is actually to put detailed conditions everywhere if everyone uses different mix? You either have a "good" mix, that will with a decent primer pair work generaly over a large set of conditions, or you have a mix/polymerase that needs a lots of optimizing.
Detailed conditions wouldn't help you in either case. 

So at the end I only divided the amplicons on "easy" and "difficult" amplification, and put there some rough Tms of the primers and amplicon lengths. For those difficult templates I tried to assess the reason for dificultness (high/low GC, ....) and some recommmended PCR aditives. It would be pointless to list all the different protocols.

 

Today many mixes may be hotstart with different conditions, of specilized polymerases also with different conditions.. you always need to use basic info (Tm from a calculator you are used to, some companies have their own, amplicon length) and use it in accordance with your polymerase recommended protocol.

(for Phusion I would add, that is more sensitive to right Tm than other polymerase I use (QIAGEN HotSTarTaq) and also, NEB has it's own Tm calculator for Phusion, that often calculate different Tm, then primer3 for example, but their works better for Phusion, of course) 


Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon


#8 meedomada

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Posted 02 May 2015 - 08:02 AM

Great Thanks  phage434 and Trof.

 from what I understood

I need optimisation which depends on the Tm as well as the polymerase used as well as the Master mix.

And since I use a master mix  wit Taq in it, I should log in to the company website and use info from it to find the conditions, is that right?

OR just optimise with different annealing temperatures.



#9 phage434

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Posted 02 May 2015 - 08:22 AM

Read your insttruction sheet for your master mix of choice.  Likely, the cycles with Taq will be something like this:

 

94 for 2m

 

then, 34 cycles of:

94 30s

55 30s

72 1m per Kb of product size

 

then

72 5m

 

Taq is substantially slower than Phusion, and less thermo stable.  The denaturing temperature has to be lower, and the extension time longer.

Make sure your initial denature is long enough to deactivate any hot start antibody, if any is present.



#10 meedomada

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Posted 02 May 2015 - 08:32 AM

Many Thanks phage434 for your precious advice. I will use your recommended settings. I will also order Phusion instead of Taq for future PCR work



#11 Trof

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Posted 02 May 2015 - 01:43 PM

@meedomada, look first into you mix manual.
There are more types of hotstart enzymes, and mine for example needs 15 minutes denaturation. You can't just take and use any recommended protocol without checking with what you really have and if it would work.

 

Also, nothing against Phusion, it's very fine proofreading polymerase, but with my (Taq) mastermix I have higer yields and much less complicated optimization, so I wouldn't just switch completely to Phusion unless I don't have a reason for that.

Again, for switching to a different mix you should first have a reason why to do it.


Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon





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