@pokie: using ePCR or traditional?
for DNA analysis will you be running the DNA through a next gen sequencer or will you be doing the good ol' DNA gel electrophoresis?
so in layman's terms you are trying to find the coding in the DNA that determines the sex of the plant by amplifying it through cycles?
The PCR product will have to be cloned into a plasmid for sequencing to occur. The PCR method is using RAPD primers which are short primers that will bind multiple places in the genome. In the paper, a single primer gave this "male specific band" thus precluding sequencing off the PCR product. Cloning a PCR product can be done via a number of ways, none of which are cheap. I will probably use the Gibson assembly or one of the PCR topoisomerase plasmids from Invitrogen. Once the PCR piece has been cloned into the plasmid, the DNA insert can be sequenced directionally. We don't sequence in house. The company that I like to use probably uses the traditional Sanger method.
If there is no band to be seen, and the paper is a fluke, this will require more effort on my part as I will have to do some bioinformatics.
sanger's method sounds definitely like a pretty penny, but it offers the best accuracy in reasonable time.
so basically, if I follow, you plan on replicating the entire genome to find that specific male sequence. would you then isolate and PCR that sequence to utilize as a future template? is it possible to create restriction enzymes that recognize that sequence so you dont have to repeat the sanger process again? i could see you utilizing that template as a standard to run against future inquiries. isolate DNA from new nep sample, run restriction enzymes, purify, PCR, then run a gel so you wouldn't need to sequence any longer to determine the sex.
14 samples already @ roughly $2400 a pop.
I had placed this pet project on the level of weekend grocery shopping.......
that high? this seems like wipe-up-a-spill type of stuff to me...
Im looking for a female for sexing experiment