Protocol for Enhancing PCR of Very Difficult Regions

Protocol for Enhancing PCR of Very Difficult Regions

Ziyun Yao, Shaoping Lin, HongMin Wu and B.A. Roe

02-14-2002

 

Target DNA (~5 ng/ul)                                                                                                            3ul

GeneAmp 10x PCR buffeundefined                                                                                                  10ul

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

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

Igepal CA-630 (1% v/v with sterile double distilled water~undefined*                                            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.

 

~undefined(500 mM KCl, 100 mM Tris-HCl, pH 8.5 in sterile double distilled water or Applied Biosystems 58002026-01)

 

~undefinedThe_7~Fdeaza_dGTP_~ARoche_part_No.92284126~B~E_dATP~E_dCTP~E_dTTP_~Athe_latter_three_are_from_Amersham~FPharmacia~B_mix_has_each_dNTP_at_a_final_concentration_of_2.0_mM_and_contains_5_mM_Tris~FHCl~E_pH_8.0_and_0.1_mM_EDTA_in_sterile_double_distilled_water.~K~Hdiv~M~2~1~0~Kdiv~M~2~1~0~0 

~undefined*_Polyoxyethylenesorbitan_monolaurate_~A1~7_v~Hv_Tween_20~E_Sigma~FAldrich_~5_P~F9416_with_sterile_double_distilled_water~B~E_Igepal_CA~F630_~A1~7_v~Hv_Octylphenoxy~Bpolyethoxyethanol_~Pwhich_is_identical_to_NonidetP~F40~E_a_nonionic_detergent~Q~E_Sigma~FAldrich_~5_I~F8896_with_sterile_double_distilled_water~B_~Aboth_detergents_are_added_to_a_final_concentration_of_0.1~7~B~E_proline_~ASigma~FAldrich_~5_P~F5607~B_and~Hor_MMNO_~A4~FMethylmorpholine_N~Foxide~B_~ASigma~FAldrich_~5_22428~F6~B_~Awhere_proline_and_MMNO_are_at_a_final_concentration_of_from_0.4M_to_0.8M~B_may_facilitate_reading_through_G~HC_rich_regions_both_in_PCR_and_sequencing_reactions. See Life Technologies European Patent Number WO09946400.