Conditions for Gonadotropin locus (Gonadotropin II b subunit or GTH2b)
Outside primer sequences: Inside primer sequences: (second round)
GTH2b-F1: 5'-cat tgt cta gga cac tca c-3' GTH2b-F1a: 5'-gtc act ctc ctc ttc acc g
GTH2b-R1: 5'-tag tac tct ttg ggt aat gc-3' GTH2b-R1a: 5'-ggc tgc agg ctc tcg atg g
Initial denaturing: 1 cycle at 95O C for 2 minutes, followed by
Denaturing,: 35 cycles of: 95 O C for 15 seconds,
Annealing: 54 O C for 40 seconds (varies for other loci-),
Extension: and 72 O C for 1 minute 20 seconds
Final extension: 72 O C for 5 minutes.
Hold at 4 O C (if desired – not necessary).
Expected Size of PCR product = 1619 bp (after first round) and 1565 (after second round) (See Fig. 1 from protocol, also below)
BstN1 is the restriction enzyme used for this locus. It works best at 60oC (at least 1 hr.), but will do fine if reactions are allowed to go overnight at 37o.
BstN1 cuts for A and B alleles: (note: asterisked numbers are lengths after 2nd round of PCR)
226 (201)* 838 329 226 (199)*
A: - + +
B: + + +
So, your gel would look like this:
AA AB BB
1000 - 1064
250 - 226 (201/199)
Restriction Digest of Gonadotropin PCR product:
Per tube e.g. x 10 (ml) (ml) ddH2O 13.5 130 10X Buffer 2 20 BstN1 0.5 5 155 (enough for 9 reactions) DNA (not in cocktail!) 4
INCUBATE AT 60o C for 60 to 90 minutes. (NOTE: You can incubate at 37o C but reactions must be allowed to go overnight)