Protocol for Enhancing PCR of Very Difficult Regions£¬ÔöÇ¿ÐÍPCR·½·¨À©ÔöÄѶȽϴóµ

Target DNA (~5 ng/ul) 3ul

GeneAmp 10x PCR buffer* 10ul

25 mM MgCl2 (Applied Biosystems 58002032-01) 10ul

7-deaza dGTP (Roche part No.92284126), dATP, dCTP, dTTP mix** 10ul

Primer pair (from mermade)(5 ul, i.e. 500 pmoles of each primer) 10ul

Polyoxyethylenesorbitan monolaurate (1% v/v with sterile double distilled water)*** 10ul

Igepal CA-630 (1% v/v with sterile double distilled water)*** 10ul

Taq polymerase (~10U/ml) 1ul

Sterile double distilled water 36ul

Final volume 100ul

Thermocycling reaction conditions:

95 degrees C for 5 minutes, followed by:

35 cycles of:

95 degrees C for 1 minutes

50 degrees C for 2 minutes

72 degrees C for 3 minutes

72 degrees C for 10 minutes

4 degrees C hold

Then, clean the PCR product containing 7-deaza-dG by adding 5 ul of SAP and 5 ul of ExoI, incubating at 37 deg C for 30 minutes followed by 80 deg C for 15 minutes. Store frozen and use 4 ul of this SAP-ExoI cleaned product in each subsequent sequencing reaction.