Primer Design

Primers sequences and sometimes stocks already exist for some genes: topA

PCR reaction

Check PCR and Quantify DNA Yields

Run 2-5 µl of sample + 2-5 µl of glycerol load buffer on a 0.8-2% agarose gel.

Load 5 µl of 0.1µg/ml DNA ladder. Use either 100-bp or 1-kb DNA ladder size as appropriate.

Ladders commonly used in lab.

Link	Company	Description	Cat #	Unit	Price
	New England Biolabs	100-bp ladder	N3231L	500 gel lanes	$212

Estimate band concentrations by eye or by saving the image in TIFF format and finding band densities with ImageJ. Compare to a reference band of similar density and determine the concentration of the original sample.

DNA purification

Use illustra™ GFX™ PCR DNA and Gel Band purification kit or Qiagen kit or similar reagent. Be sure to elute in buffer compatible with DNA sequencing applications. (Buffer 6 for the GE kit, or ddH₂O)

For all practical purposes assume 90% recovery of the input DNA sample as long as it is >200 bp in length. Smaller fragments are not bound as efficiently by the column.

Sequencing Reaction

Normal submissions to RTSF at MSU should be in 96-well plates.