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

PCR conditions:

Initial denaturing: 1 cycle at 95^O 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 60^oC (at least 1 hr.), but will do fine if reactions are allowed to go overnight at 37^o.

BstN1 cuts for A and B alleles: (note: asterisked numbers are lengths after 2^nd round of PCR)

226 (201)* 838 329 226 (199)*

A: - + +

B: + + +

So, your gel would look like this:

SCHEMATIC:

GTH2b/BstN1

AA AB BB

Band sizes

1500 -

1000 - 1064

838

750 -

500 -

329

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                

TOTAL:                          20

INCUBATE AT 60^o C for 60 to 90 minutes. (NOTE: You can incubate at 37^o C but reactions must be allowed to go overnight)