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Real Time PCR product size

Real Time PCR product size - PCR - Polymerase Chain Reaction Forum

Real Time PCR product size - PCR - Polymerase Chain Reaction Forum. Discuss and ask questions about PCR troubleshooting, PCR protocols and methods, PCR products, and PCR theory.


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  #11  
Old 02-23-2010, 02:58 PM
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Default Re: Real Time PCR product size

Isabel,
Those thermal cycling conditions won't work with those primer Tms. You'll never achieve efficient primer hybridization. I would redesign the primers to have Tms of 58oC -- 60oC. If you have access to Primer Express software I'd use that for the design. You could also try optimizing the primer concentrations to fit the primers you've listed. This is generally not the norm with Taqman though.
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Old 09-13-2010, 10:37 AM
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Default Re: Real Time PCR product size

Hi,

I'm starting to do real-time PCR now and I'll need to amplify 550-560bp products. I know it's not ideal but there's no way I can do it with smaller amplicons. Can someone give me some hints so that I can have a starting point from which I can optimize my reactions? Is SYBR Green the way to go? Thanks in advance.
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  #13  
Old 09-14-2010, 05:14 PM
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Default Re: Real Time PCR product size

If you can't get shorter amplicons start with what you've got. SYBR Green I based assay is a good way to begin with. For start I would use the standard protocol - you should get one with your SYBR Green reaction kit. Just follow the instruction and see what you'll get.

Hybridization temperature may be crucial here, as well as elongation time.

Later on you're almost certainly going to do some adjustments.
First you need to make sure you have your desired product though and that there's high specificity of your reaction. Surely you'll need to start your assays with your positive control. With long amplicons don't expect perfect efficiency of your reactions but make sure primers and Mg concentration gives you maximal efficiency you can get with the lowest primer-dimers formation. It would be ideal if there weren't any primer-dimers with your positive control. They can sometimes occur with negative control with SYBR Green I though and then make sure if there are only primer-dimers.

Have a go with it and check what you'll get.
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  #14  
Old 05-11-2011, 12:49 AM
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Default Re: Real Time PCR product size

Not trying to say anyone else is wrong here but I will give you some anecdotal evidence from my own experience with qPCR. I have assayed genes with pcr products up to 485bp with no problems. If you are using relative quantification, it doesn't really matter if your pcr efficiency is 99% or 70%. What is critically important is that all your primers have similar efficiency. So of course you need to know the efficiency of each primer relative to the other ones that you are using, but in theory you don't even need to know the value of their efficiencies. The only other point is for people using SYBR. Since SYBR molecules bind to double stranded nucleic acid, the pcr product's fluorescence will be proportional to its length because more SYBR molecules can bind to the product the longer it is. However since you will be normalizing everything to another gene, this difference shouldn't be large enough to make any difference to your results.
So overall it doesn't really matter how long your product is as long as it gets produced in the time you allow for extension. I use 45 seconds at 72 degrees with Fermentas Maxima Taq and have no problem at all generating a ~500bp product.
If someone can give me good evidence that what I've said is wrong somehow please say so!
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  #15  
Old 06-10-2011, 12:16 AM
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Default Re: Real Time PCR product size

This is all dependent on what type of analysis you intend to do, to tell you the truth.

A. Are you looking to do simple absence or presence ? IF so Sybr is just fine and you can simply shotgun it with a touchdown PCR if you don't know your annealing temps... just make sure you take your BP amplicon and divide this by 25 to reach your annealing hold time.

B. Are you doing relative Quantification? This is a loaded question because there are two types of RQ. The first type as listed above RELIES on perfect efficiency which means that with large amplicons like this, you better be 1. God's gift to QPCR, 2. come with natural ability that makes Tiger Woods cry, 3. Normal like the rest of folk, Better use the other relative quant and not this method. If you are #3, then I would suggest running a housekeeping gene standard curve and a target gene standard curve, which will then be your normalizing factor for all Cp values after this point. But enough of that!

3. Genotyping ; melting curves, melting curves, melting curves. This is relatively easy as you can simply add either a Tm melt or an HRM analysis at the end of your sybr green run. This is also another option for presence or absence.

4. Basic Quantitation : You can do Sybr Green but you can also run the probes method which I personally do, but you would need some extra $$ initially. Either way, just be sure to run a Standard curve - you could run a 10x starting from 100ng, 10ng, etc. This will also establish your efficiency and linearity. Be sure your professor/boss isn't watching at this point because this also shows just how wonderful or stupendously bad your pipetting technique is.

Have fun!
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  #16  
Old 07-19-2012, 08:05 AM
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Default Re: Real Time PCR product size

Quote:
Originally Posted by Aurora Borealis View Post
I think it depends on your PCR and what you're looking for. We can't use large products, the sensitivity gets worse in our PCR.
Yes i agree with you!!!
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Old 12-19-2012, 11:50 AM
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Default Re: Real Time PCR product size

The Applied Biosystems 7500 Fast Real-Time[Only registered users see links. ]System offers maximum performance in the minimum time. Fully optimized for Fast cycling, the 7500 Fast delivers high-quality results in as little as 30 minutes.
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  #18  
Old 01-15-2013, 05:11 AM
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Default Re: Real Time PCR product size

[Only registered users see links. ] For a real-time PCR with SYBR natural, PCR items should in organize of 150~80bp. More time item might work, but eat PCR element more easily than brief one, outcome in shorting the variety of rapid stage in boosting, anyway, a 300bp items seems still appropriate for real-time PCR with SYBR natural.
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  #19  
Old 01-23-2013, 07:17 AM
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Default Re: Real Time PCR product size

[Only registered users see links. ]Real-Time PCR System combines all of the qPCR features you want in a single high performance instrument, so that you can optimize your research productivity. With a simplified workflow, intuitive software, touch-screen interface, and one-button protocols for error minimization, the ViiA™ 7 System offers exceptional reproducibility with minimal well-to-well and instrument-to-instrument variation.
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  #20  
Old 01-31-2013, 05:07 AM
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Default Re: Real Time PCR product size

[Only registered users see links. ]Optimal amplicon size in RealTime PCR is about 150bp, although new kits can handle products as large as 400bp (FullVelocity SYBR Green - Stratagene, DyNamo SYBR Green - Finnezymes). I had a 350bp amplicon and results were just fine. But still you have to keep in mind that larger products may strongly affect efficiency of RealTime.
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