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

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

4 µl of [PCR product + water] that has a total of3-10ng of DNA

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

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

  

Many of you will have a band equally bright to the 500bp SSband, 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 ascompared to the concentration required per microliter(#6)?    [For example, if you had a sample that had1500ng DNA/µl and you needed ~125ng DNA/µl, your sample would be12x 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. Thisdiluted DNA would need to be mixed in an empty, separate tube andcan be at any volume so long as the ratio is correct (though keepin mind you only have ~40 µl of PCR product remaining).

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

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