How easy is to distinguish strains using RFLP? Is it straightforward? where can I find the protocol?
Is there an easier method to distinguish between them?
Thank you
I’m assuming you mean strains not carrying mutations that result in a visible phenotype?
A good place to start with questions like these is often Wormbook. Other, somewhat different protocols (such as this one ) can be found easily using Google.
Using the protocol in the Wormbook chapter as a starting point, there are some points where cost can be reduced and time can be saved:
(1) The protocol asks for 5 units of Taq polymerase per reaction; this can be cut at least 8-fold, and probably more. One way to use less Taq is to denature at slightly lower temperature (94 or 94.5), as Taq polymerase has a half-life of 5 minutes at 95 degrees. The primer concentration can also be halved.
(2) The denaturation, annealing, and especially extension times are all longer than required (this mostly saves time, but the first of these can save on Taq polymerase).
(3) As you can see from the other linked protocol, lysate from a single worm can be split for use in several PCR’s; I’ve never tried splitting into 40, as they recommend, but I haven’t had problems splitting lysate 6 or 7 ways. I usually lyse in 10 ul 1x lysis buffer and dilute the lysate with water when making the PCR reactions.
(4) Also as is indicated in the other linked protocol, the digest can usually be done by adding a mix containing 10x enzyme buffer, enzyme, and BSA if requested directly to the PCR product, for a final concentration of 1x restriction buffer + ~0.8x Taq buffer, and incubating for a few hours. Obviously, this isn’t quite the recommended buffer, but most digests work anyway.
Yes, I need to distinguish between two wild isolates with no obvious individual phenotype to discard a inter-strain contamination issue.