I want to create my own DNA ladder for gel electrophoresis in order to save money. Has anyone any recommendations on particular enzymes to use or the most validated methodology? We have run a few trials here in our laboratory however I would like to see how others have faired using their sequences.
Thanks
I would suggest that you buy mealworm beetle larvae (Tenebrio molitor) from a pet shop (very cheap). When back to the lab, snap-freeze them in Liquid nitrogen, grind them...phenol-extract DNA (according to Maniatis) and digest it with EcoR I you will obtain a multimer of 142 bp ladder that you can use it as a marker (very abundant satellite DNA, I think more than 30% total DNA/google it to be sure) . Please you can only use it in agarose gel because if I remember well, due to its intrinsic curvature its migration in native PAGE is altered. I’ve done it almost 16 years ago.
all of the companies have plasmids that, when digested with a restriction enzyme, give you the size pattern. Maxi preps can be cheap if you don't use a kit. restriction enzymes can be cheap if you use EcoRI or other commonly used enzymes.
I would suggest that you buy mealworm beetle larvae (Tenebrio molitor) from a pet shop (very cheap). When back to the lab, snap-freeze them in Liquid nitrogen, grind them...phenol-extract DNA (according to Maniatis) and digest it with EcoR I you will obtain a multimer of 142 bp ladder that you can use it as a marker (very abundant satellite DNA, I think more than 30% total DNA/google it to be sure) . Please you can only use it in agarose gel because if I remember well, due to its intrinsic curvature its migration in native PAGE is altered. I’ve done it almost 16 years ago.
Nice timing. We just launched the world's first open source DNA ladder plasmids. These let you make your own ladder with even spacings, using the cheap EcoRI enzyme Colin mentioned. http://www.geneandcell.com/collections/open-source/products/dna-ladder-plasmids
If you are using wild-type lambda DNA, Digest with EcoRI and digest another sample with HindIII. You can mix them together or use them separately in two lanes of your gel. You get a nice banding pattern with these two enzymes. You can also try BstEII, which also gives a nice pattern. Remember to incubate your ladder at 65°C for a few minutes before loading to denature the cohesive ends.
- Badges
- Science method