M13 amplification procedure

PCR mastermix

1. Use the cycling conditions labeled as M13 (under Lab or Martin) on Hulcr lab thermocycler.
2. For extra care (ie publications), prepare a replicate PCR for each extraction.

Running the gel

1. Prepare a 1% agarose gel (careful, it will be fragile). Make sure the gel is level when pouring!
2. Use extra reagents, but make sure the volume will fit in the comb you use for making the wells:

1. When loading, arrange samples from similar morphotypes together. If replicate PCRs were prepared, do not group them directly next to corresponding replicate sample.
2. Place both a 100bp and 1000 bp ladder on either side of every 5-10 reactions ran. This will aid in reading the gel, and will help to show if the DNA is moving at different speeds in different parts of the gel.
3. Run for up to 3 hours at 100-110V. Check every 45min-1 hour and take photo. The longer you run the gel, the more differentiated the bands will become, making it easier to read.

Reading the gel:

1. Use a photo editor (ie photoshop) to adjust the contrast/brightness so that all bands can be seen and differentiated between samples.
2. Use a line tool in the photo editor to connect bands in ladders with the same lengths. Use the slope of these lines to gage the distances traveled between samples.
3. Note any differences in fragment location and presence/absence between samples. Same species should be basically identical in their arrangement. Samples that appear different should be sequenced at a variable locus for confirmation.