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  • Pooling pcr, quantitate pcr, equimolar ratios

    Hi,
    I need urgent help. This is my first time working on NGS samples and quantitating pcr products (not dna extracts per se).

    I will be submitting my samples (long-pcr products) for NGS sequencing this week. However, I need to figure out how to quantitate my pcr product and combine/pool them in equimolar ratios. How do I do this if I do not use qPCR and Bioanalyzer?
    I have 2-4 different pcr products for each species (20 species total). Each pcr product has different sets of primers that are overlapping. How do I combine them so that I end up with only 1 tube of sample for each species?
    Do I just pour them in 1 bigger tube? e.g. if I have 25ul pcr, combining 2 of them will give me a 50ul total pcr product?
    I was advised to run a gel and compare the brightness of the bands; and if they have the same brightness, it would be okay to just combine them. Is this okay? I am not confident in trusting that 2 or more samples for each species have/will have same brightness if I cannot measure them or no criteria other than brightness to look at. Are there other ways of quantitating pcr products?
    How do I combine pcr products with equimolar ratios? How do I compute this?
    Thank you.

  • #2
    Fluorimetry is one option. Don't forget to adjust for your PCR product length.

    --
    Phillip

    Comment


    • #3
      Thanks, Phillip!
      But what do you mean by adjusting the PCR product length? Let's say I'm working on around 16-20Kb bp fragments...

      Comment


      • #4
        Originally posted by xet1201 View Post
        Thanks, Phillip!
        But what do you mean by adjusting the PCR product length? Let's say I'm working on around 16-20Kb bp fragments...
        ???

        Under very specialized PCR conditions you might generate 16-20 kb products, but there is zero chance you would obtain sequence from any second generation sequencer off such an amplicon. So this is a typo?

        --
        Phillip

        Comment


        • #5
          Given that you have at most 1.6 Mb to sequence (4*20*20kb) I dont think you have to care too much about getting equimolar ratios. I lane of 2x100 bp reads would give you > 2000 fold coverage on average.

          Comment


          • #6
            DNA quantitation and pooling

            Originally posted by xet1201 View Post
            Thanks, Phillip!
            But what do you mean by adjusting the PCR product length? Let's say I'm working on around 16-20Kb bp fragments...
            Let's hope you're not really submitting DNA of this length...
            Fluorimetry methods like Quant It/ Pico Green measure DNA concentration by mass. so if you have a 200 bp PCR product and a 400 bp product, and the same molar concentration of each, the 400 bp fragment will have double the signal. what you need to do is quantitate by mass in a fluorimeter or fluorescence plate reader, then convert the results to molar, then pool.

            But as was mentioned, you'll likely get plenty of data so if you run the gel and just make sure you have a decent amount of PCR product you should be OK.

            But if those fragments are 16-20k you will get very few results. Ask your sequencing core what kind of sequencer they're using and the recommended fragment length.

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            • #7
              Hi everyone,

              I have a basic question for pooling of barcoded amplicons. I have 30 samples that I have extracted DNA from and will be amplifying with barcoded primers for Illumina sequencing. My DNA extracts are fairly low in DNA concentration (e.g. ~2-5ng/ul). I have been processing a "test" sample of similar origin and quality through what I hope to do for my actual samples.

              Thus far, I have amplified the region I am interested in, and the product ends up being ~420bp. I took that product and did a PCR cleanup and size selection. I then quantified this product via Qubit and got an estimated concentration of 5ng/ul in 20ul. If I have done my math right, this equates to:
              ((5.0)*(1000000)*(1/660)*(1/420)) = ~18nM (this means 18nM of product per ul?)

              Now, when I do this same process with my actual samples, I will then need to pool the samples at equimolar concentrations and then further dilute that down eventually to 10nM for sequencing. So, if say I did the quantification of all my size-selected and purified products and got a range of values, maybe 3nM, 5nM, 4nM (lets just use 3 for an example). Would it then be correct to take 10nM of each and pool? So, 3.33ul of the 3nM, 2ul of the 5nM, and 2.5ul of the 4nM? This would give me a 30nM pooled library that I could then dilute down to 10nM?

              Sorry if this is a bit of a basic question. I have worked with prepping samples for barcoded sequencing in the past, but my samples were part of a larger run that was pooled for us after we provided the DNA concentrations.

              Thanks in advance for any help/advice you may have!

              Comment


              • #8
                Or, perhaps I am getting really confused.

                If instead I looked at the ng/ul concentrations of each sample and had the following concentrations:
                Sample A - 5ng/ul
                Sample B - 1ng/ul
                Sample C - 3ng/ul

                I could use the following volumes and load 20ng of each sample.
                Sample A - 4ul
                Sample B - 20ul
                Sample C - 6.6ul
                Which would be 60ng in 30.6ul or 1.96ng/ul. Then, if I use the formula I mentioned previously and converted this to nM would be ~7nM. Even though these numbers are low, is this right?

                And, if my concentrations were that low, could I ethanol precipitate the pooled library and re-suspend it in a smaller volume to increase the nM concentration?

                Thanks again, hoping to get this straight in my head.

                Comment

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