Question

The second PCR you perform requires that only 3-10ng of PCR product be used for ideal results. This, of course, must be paired with careful lab technique, such as keeping the sequencing PCR mix ice-cold and not contaminating the reaction with outside sources of DNA such as that on your fingertips. The following steps will take you through a proper dilution of the PCR product so that you get the appropriate concentration of DNA for your sequencing reaction. Remember: you cannot pipet 0.5 µl or 1 µl with a P20 micropipettor. The lowest volume you can pipet is 2 µl. Attempting to set it lower than that can break the equipment!

As per the instructions on page 35 of your manual, you need to add:

4 µl of [PCR product + water] that has a total of 3-10ng of DNA

to the 0.2ml PCR tube that already contains 6 µl of the Big Dye Master Mix (=Sanger Sequencing PCR Mix) to get a 10 µl reaction.

If you need 3-10 ng/4 µl, what is the DNA concentration range per 1 µl required?

  

Many of you will have a band equally bright to the 500bp SS band, so go back to #3 in Step 1 and record that amount here:

______________ ng DNA/ µl PCR product

How many times higher is the concentration of DNA in #7 as compared to the concentration required per microliter (#6)?    [For example, if you had a sample that had 1500ng DNA/µl and you needed ~125ng DNA/µl, your sample would be 12x higher than it should be.]

To get DNA at the volume and concentration you need, use the # calculated in #8 to determine your proportion of sample:water. This diluted DNA would need to be mixed in an empty, separate tube and can be at any volume so long as the ratio is correct (though keep in mind you only have ~40 µl of PCR product remaining).

Using the example from #8, if we had a sample that was 12x more concentrated than required, we would make a dilution that was 1/12 sample and 11/12 water. This would be a 1:11 ratio. Since the minimum volume we can accurately pipet is 2µl, we could make a tube with 2µl sample (=3000ng DNA) and 22µl water = total of 3000ng DNA/24µl water = 125ng/µl.   4µl of this diluted DNA could then be pipetted out and added to the desired reaction.

Work with your partner to determine how to make a DNA dilution that would be appropriate for sequencing, yielding at least 4µl total volume containing only 3-10ng DNA. Again, your minimum pipetting volume is 2µl.   

Answer 1

For 3-10 ng in 4 microliters, we need the concentration range of 0.75 ng/microlitre (3ng/4 microlitre) - 2.5 ng/microlitre(10 ng/4microlitre). [Formula 1: Concentration = weight in ng / volume in microlitre]

We need the above-stated concentration. So we can dilute our sample according to S₁V₁=S₂V₂

For a minimum concentration of 0.75ng/microlitre

Now, since the concentration required is very less what we can do is prepare 40 microlitres of 0.75 ng/microlitre of DNA sample from PCR1 product and then pipette out 4 microlitres from the solution. We had to do this because the minimum pipetting amount is 2 microlitres and any concentration larger than 1.5 ng/microlitre couldn't be diluted using P20 because of measurement constraints.

Hence to prepare 40 microlitres of 0.75ng/microlitre concentration

0.75*40 = x*y, where x is the concentration you obtained in PCR1 product and y is the volume of the PCR product you would take to be diluted to 40 microlitres with double distilled water (molecular grade, nuclease-free)

For 2.5 ng/microlitre (i.e. 10 ng in 4 microlitre) strength replace 0.75 with 2.5 in the equation above.

If the problem still persists please post the question with the concentration of PCR1. You can calculate the same using Formula 1.