sense/antisense specific pcr - design of sense/antisense specific pcr primers (Feb/10/2005 )
I'm a biotech honours student currently working on breeding 2 cultivars of transgenic medicago, one with a + sense viral insert, and one with a -sense. I'd like to desing a PCR test to determine in the plants, and in their prodgeny, which of these inserts are present (I'm looking for presense of the inserts, not expression at this stage).
The inserts were originally transformed into a B35S BgN plasmid (a few years ago, not by me!.. im just picking up where they left off), which was then subsequently transformed into a Bin19 plant transformation vector. Since i don;t have an exact sequence of the whole construct, i was thinking i could use 2 pairs of primers: one common primer used in each test, with +sense homology to part of the upstream of the insert, and 2 unique antisense primers, one each for sense and anti-sense ...
i.e. in the plant..
Bgn/p19 +/- sense insert
(primer1) (primer 2 or 3)
sorry. . . my formatting got all out of whack...
the ---- bits are BgN/Bin19, |||| bits are the +/- sense insert...