Ziyun Yao, Shaoping Lin, HongMin Wu and B.A. Roe
02-14-2002
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.
*(500 mM KCl, 100 mM Tris-HCl, pH 8.5 in sterile double distilled water or Applied Biosystems 58002026-01)
**The 7-deaza dGTP (Roche part No.92284126), dATP, dCTP, dTTP (the latter three are from Amersham-Pharmacia) mix has each dNTP at a final concentration of 2.0 mM and contains 5 mM Tris-HCl, pH 8.0 and 0.1 mM EDTA in sterile double distilled water.
*** Polyoxyethylenesorbitan monolaurate (1% v/v Tween 20, Sigma-Aldrich # P-9416 with sterile double distilled water), Igepal CA-630 (1% v/v Octylphenoxy)polyethoxyethanol [which is identical to NonidetP-40, a nonionic detergent], Sigma-Aldrich # I-8896 with sterile double distilled water) (both detergents are added to a final concentration of 0.1%), proline (Sigma-Aldrich # P-5607) and/or MMNO (4-Methylmorpholine N-oxide) (Sigma-Aldrich # 22428-6) (where proline and MMNO are at a final concentration of from 0.4M to 0.8M) may facilitate reading through G/C rich regions both in PCR and sequencing reactions. See Life Technologies European Patent Number WO09946400.
