PCR Amplification from Genome

1. Extract template M. xanthus DNA with genomic DNA miniprep kit or other protocol. Dilute 1:10 to use for this PCR protocol (you will receive already diluted DNA). Note: check freezer first we probably have some isolated gDNA.

2. Prepare PCR reaction master mix. Calculate for the number of reactions plus 1. You will run your experimentalsamples w ith M. xanthus template DNA and your designed primers, a positive control with M. xanthus template DNA and 16s rRNA primers, and a negative control with your designed primers but no template DNA. Keep all your tubes on ice.

Ex. if you have 4 experimental samples, you need 4, plus one for positive control, one for negative control, and add one to account for some loss due to pipetting. You would calculate your mastermix for 7 reactions.

To a 1.5ml centrifuge tube, add: (multiply volumes by #samples + 1)

13.5 uL PCR Water 5.0 uL GoTaq Buffer 1.5 uL 25mM MgCl2 1.25 uL DMSO 0.5 uL dNTPs 0.25 uL Taq Polymerase

3. Aliquot 22 μl of the master mix into labeled PCR tubes (these are smaller than the regular epi tubes) for each reaction you are running. You can number them as long as you immediately include the label and corresponding sample in your lab notebook.

4. To the experimental tubes and the positive control tube, add 1 μl of M. xanthus template DNA, and to the negative control add 1 μl of PCR grade water.

5. Add 1 μl each of your 10uM stocks of your chosen forward and reverse primers to and experimental PCR tubes so that the total volume in the tube is 25 μl. Add 1ul of the 16s rRNA control primers to the positive control tube and to the negative control tube. Tap tubes or briefly centrifuge if possible to pull any droplets to the bottom of the tube. For now, you will not run a negative control for every set of experimental primers, but this is something you can do if you get unexpected bands to make sure primers are not contaminated.

6. Group your samples based on annealing temp and extension time. The 16S rRNA control primers have an annealing temp of 65C and an extension time of 30 sec and will likely need to go in its own machine. Talk to Jess about using a pre-programmed PCR machine in superlab.

|Initial Denaturation 95C 3 min | | | Denature 95C 30 sec | 30x Anneal calculate 30 sec | | Elongate 72C calculate | |Final Elongation 72C 1 min |Hold 4C indefinite

7. Store at -20C until used for ligation.