Advantage
®
-HF PCR Kit
User Manual
(PT3139-1)
Catalog #K1909-1, -y
Storage conditions: –20
°
C
FOR RESEARCH USE ONLY
(PR76834)
Page 1: ...Advantage HF PCR Kit User Manual PT3139 1 Catalog K1909 1 y Storage conditions 20 C FOR RESEARCH USE ONLY PR76834...
Page 2: ...opment activities of the purchaser when this product is used in conjunction with a thermal cycler whose use is covered by the up front fee component Rights to the up front fee component must be obtain...
Page 3: ...e and TaqStartTM Antibody to provide a superior level of specificity Advantage HF thus combines the benefits of Pfu and the Advantage Enzyme System to deliver a high fidelity enzyme system The HF Adva...
Page 4: ...s after 25 PCR cycles b agrees with published data Ling et al 1991 Cariello et al 1991 High fidelity amplification of cDNA and Genomic templates Advantage HF was used to amplify several cDNA templates...
Page 5: ...elongation efficiency by combining the HF and cDNA buffers in varying proportions Figure 4 Panel A shows the fidelity resulting from the use of varying percentages of the HF Buffer see p 3 for descri...
Page 6: ...PCR TaqStart mediated hot start PCR has been shown to significantly improve the efficiency and specificity of DNA amplifications Kellogg et al 1994 CLONTECHniques April 1994 Antibody mediated hot sta...
Page 7: ...g l in the following storage buffer Concentration 1X in 50X Component Concentration 50 Glycerol 1 0 40 mM Tris HCl pH 7 5 0 8 mM 50 mM KCl 1 0 mM 25 mM NH4 2SO4 0 5 mM 1 mM EDTA 20 M 5 0 mM mercaptoe...
Page 8: ...ma Cat M 3516 0 5 ml PCR reaction tubes We recommend Perkin Elmer GeneAmpTM 0 5 ml reaction tubes Cat N801 0737 Thermal cycler Perkin Elmer DNA Thermal Cycler 480 9600 or equivalent Dedicated pipettor...
Page 9: ...esign will vary from one application to the next In general however primers should have a Tm of at least 70 C to achieve optimal results in a two step cycling program with a 68 C annealing extension s...
Page 10: ...several times c Use a Master Mix Using a Master Mix greatly reduces tube to tube variation There fore use a Master Mix whenever you set up multiple PCR reactions If multiple templates are being tested...
Page 11: ...a 0 5 ml PCR tube Positive Negative Control Control 32 l 37 l Purified H2O 5 l 5 l 10X HF PCR reaction buffer 5 l Control DNA template 0 2 ng l 2 l 2 l Control primer mix 10 M ea 5 l 5 l 10X HF dNTP m...
Page 12: ...68 C for 3 min d Soak at 15 C Soak at 15 C a 25 cycles for multiple copy genes or medium to high abundance cDNAs 30 35 cycles for single or low copy number genes or rare cDNAs For most applications we...
Page 13: ...ry in a 50 l PCR reaction containing 4 l of 10X HF Buffer 80 final and 1 l of 10X cDNA Buffer Optimalconditionsfortheamplificationofotherfragmentsshould be determined individually We recommend initial...
Page 14: ...e in increments too high of 2 4 C Suboptimal primer Redesign your primer s after confirming the accu design racy of the sequence information If the original primer s was less than 22 nt long try using...
Page 15: ...orethan5mM thiscanreducetheeffective concentrationofMgtobelowaminimumlevel Increas ing the concentration of Mg2 can result in lower fidelity dNTPs not optimal The Advantage HF PCR Kit contains a caref...
Page 16: ...ecrease the extension time in 1 2 min increments too long Poor template Check template integrity by electrophoresis on a de quality denaturing agarose gel Repurify your template if necessary Touchdown...
Page 17: ...t PCR analysis in separate laboratory areas with separate sets of pipettors Laboratory benches and pipettor shafts can be decontaminated by depurination Wipe surfaces with 1N HCl followed by 1N NaOH T...
Page 18: ...91 Touchdown PCR to circumvent spurious priming during gene amplification Nucleic Acids Res 19 4008 Frey B Suppmann B 1995 Demonstration of the ExpandTM PCR system s greater fidelity and higher yields...
Page 19: ...ntage Genomic PCR Kit K1906 1 y Advantage Genomic Polymerase Mix 8418 1 Advantage GC cDNA PCR Kit K1907 1 y Advantage GC cDNA Polymerase Mix 8419 1 Advantage GC Genomic PCR Kit K1908 1 y Advantage GC...
Page 20: ...Marathon ReadyTM TaqStartTM and TthStartTM are trademarks of CLONTECH Laboratories Inc GeneAmpTM is a trademark of Hoffmann La Roche Inc Deep VentR TM is a trademark of New England Biolabs Inc 1997 CL...
