Transcription of PrimeSTAR® Mutagenesis Basal Kit
1 R046A PrimeSTAR MutagenesisBasal Kit v201601Da I ..3II ..3 III ..3IV ..5V ..8VI ..10 VII ..13 VIII ..13IX ..132 R046 APrimeSTAR Mutagenesis Basal Kit PCR PrimeSTAR Max dna polymerase DNA PrimeSTAR Max PCR PrimeSTAR Max 5 PCR PCR PCR PCR I 25 PrimeSTAR Max Premix 2 625 lControl template pUC118 DNA 50 ng/ l 10 lControl Primer F 20 pmol/ l 10 lControl Primer R 20 pmol/ l 10 lXho I 10 l10 Xho I buffer 10 l 6 10 10 Control template 10 100 pg/ l II 20 CIII 5 15 PCR 5 PCR 1 PrimeSTAR Max PCR PCR
2 PCR PrimeSTAR Max PCR 3 http R046A 1 PCR 5 15 PrimeSTAR Max PCR PrimeSTAR Max 5 3 PCR 2 5 A B A 5 3 B 3 5 A B 15 15 4 R046A PrimeSTAR Max Thermus thermophilus HB8 DNA 8 500 bp PCR mutation frequency PrimeSTAR Max dna polymerase 230,129 9 PrimeSTAR HS dna polymerase A High Fidelity PCR PCR Fidelity PrimeSTAR Max PCR GC 370.
3 656 4 10 -2 IV 3 A C 15 3 18 1 15 5 15 GC 75% PCR PCR 5 15 PCR PrimeSTAR MaxPrimeSTAR HSA High Fidelity Pfu DNA PolymeraseTaq DNA Polymerase5 http R046 AGAC TGA 5'-C G T C G T T T T A C A A C G T C G TG A CT G G G A A A A C C C T G G C G T T A C C C A A C-3'3'-G C A G C A A A A T G T T C G A G C AC T GA C C C T T T T G G G A C C G C A A T G G G T T G -5' 5'-C G T C G T T T T A C A A C G T C G TT G AT G G G A A A A C C C T G G C G T T A C C C A A C-3'3'-G C A G C A A A A T G T T C G A G C AA C TA C C C T T T T G G G A C C G C A A T G G G T T G -5' 15 base 5'-C G T C G T T T T A C A AC G T C G TT G AT G G G A AA A C C C T G G C G T T A C C C A A C-3'3'-G C A G C A A A A T G T TC G A G C AA C TA C C C T TT T G G G A C C G C A A T G G G T T G -5' 18 base 3' 5'- C G T C G T T T T A C A AC G T C G TT G AT G G G A AA A C C C T G G C G T TA C C C A A C-3'3'- GC A G C A A A A T G T TC G A G C A A C TA C C C T TT T G G G A C C G C A AT G G G T T G -5' 5'-C G T C G T T G AT G G G A AA A C C C T G G C G T T-3'3'-C A G C A A A A T G T TC G A G C AA C TA C C C T T-5' 1 15 3 A 5'-C G T C G T T T T A C A A C G T C G T G A CT G GG A A A A C C C T G G C G T T A C C C A A C-3'3'-G C A G C A A A A T G T T C G A G C A C T G A C C C T T T T G G G A C C G C A A T G G G T T G -5' 5'-C G T C G T T T T A C A A C G T C G T G A C G A A A A C C C T G
4 G C G T T A C C C A A C-3'3'-G C A G C A A A A T G T T C G A G C A C T G C T T T T G G G A C C G C A A T G G G T T G -5' 15 base 5'-C G T C G T T T T A C A A C GT C G T G A C G A A A A C C CT G G C G T T A C C C A A C-3'3'-G C A G C A A A A T G T T C GA G C A C T G C T T T T G G GA C C G C A A T G G G T T G -5' 18 base 3' 5'- C G T C G T T T T A C A A C GT C G T G A C G A A A A C C CT G G C G T T A C C C A A C-3'3'- G C A G C A A A A T G T T C GA G C A C T GC T T T T G G GA C C G C A A T G GG T T G -5' 5'-T C G T G A C G A A A A C C CT G G C G T T A C C-3'3'-C A A A A T G T T C GA G C A C T GC T T T T G G G-5' 3 B 6 http R046A CTCGAG 5'-C G T C G T T T T A C A A C G T C G T G A C T G G G A A A A C C C T G G C G T T A C C C A A C-3'3'-G C A G C A A A A T G T T C G A G C A C T G A C C C T T T T G G G A C C G C A A T G G G T T G -5' Insertion 5'-CG T CG T T T T A C AA CG T CG T GA C TC T CGAGGGGAAAA C C C T GGCG T T A C C C AA C-3'3'-GC AGC AAAA T G T T CGAGC A C T GA GAGC T C C C C T T T T GGGA C CGC AA T GGG T T G-5' 15 base 5'-CG T CG T T T T A C AA CG T CG TGA C TC T CGAGGGGAAAA C C C T GGCG T T A C C C AA C-3'3'-GC AGC AAAA T G T T CGAGC AC T GA GAGC T C C C C T TT T GGGA C CGC AA T GGG T T G-5' 18 base 3' 5'-CG T CG T T T T A C AA CG T CG TGA C TC T CGAGGGGAAAA C C C T GGCG T T A C C C AA C-3'3'-GC AGC AAAA T G T T CGAGC AC T GA GAGC T CC C C T TT T GGGA C CGC AA TGGG T T G-5' 5'-GA C T C T CGAGGGGAAAA C C C T GGCG T T A-3'3'-AAAA T G T T CGAGC AC T GAGAGC T CC C C T T-5' 1 15 3 C 7 http R046AV 1 10 kb 1 10 100 pg/ l 2 2 Mutagenesis PCR PrimeSTAR Max Premix 2 Primer 1 Primer 2 Plasmid 10 100 pg/ l 25 l10 pmol10 pmol1 lup to 50
5 L1 M3 PCR PCR 98 C55 C72 C10 sec .5 30 cycles 3 PrimeSTAR Max dna polymerase 3 4 4 PCR 2 l 5 7 100 l 1 cfu/ g DNA PCR cfu/ g 106 2 PCR 50 l 10 pg 100 pg DNA PCR DNA PCR 50 l DNA 100 pg 3 DNA 10 100 pg DNA 30 8 R046A 4 PCR
6 5 PCR 108 cfu/ g pUC118 6 DNA PCR PCR 7 PCR PCR 5 DNA E. coli HST08 Premium JM109 DH5 HST02 Competent cells PCR pUC118 DNA - X-Gal IPTG L-Amp Xho I 1 2 9 http R046 AVI - 1 pUC118 DNA 10 pg PrimeSTAR Max PCR 50 l 2 l E.
7 Coli JM109 3 108 cfu/ g pUC118 100 l 0 C30 42 C 45 SOC 1 ml 37 C 1 100 l LB Amp IPTG X-gal PCR 98 C55 C72 C10 cycles 1 3 1, 3 2, 6 1, 6 Control primer F Control primer R 1. pUC118 DNA 10 pg 3 99% - 2 - 1 117 117 370,656 4 PCR PrimeSTAR Max dna polymerase PCR 10 R046A - 3 kb pUC118 DNA Hinc II kb kb 100 pg PrimeSTAR Max PCR 50 l 2 l E. coli JM109 1 108 cfu/ g pUC118 100 l 0 C 30 42 C 45 SOC 1 ml 37 C 1 100 l LB Amp IPTG X-gal 10 Hinc II PCR 98 C55 C72 C10 cycles 100 pg 158 3 98% 10 kb 4A Hinc II 1 kb DNA 4B 4 A non-cut 1 2 11 M -Hind III digest M 1 2 3 4 5 6 7 8 9 10 11 M 4 B Hinc II 1 2 11 M -Hind III digest M 1 2 3 4 5 6 7 8 9 10 11 M11 R046A 5 Xho I kb kb kb kb kb kb - 4 kb kb kb pUC118 DNA 100 pg Xho I PrimeSTAR Max PCR 50 l
8 2 l E. coli JM109 4 108 cfu/ g pUC118 100 l 0 C 30 42 C 45 SOC 1 ml 37 C 1 100 l LB Amp 10 Xho I Xho I PCR 98 C55 C72 C10 cyclesX 20 kb 40 kb 60 kb kb kb kb 1,496 534 21 100% 10/10 100 10/10 80% 8/10 5 kb 100% kb 80% 12 http R046 AVII PCR 2 C 2 C 2 step PCR 98 C 10 sec. 68 C 5 10 35 40 10 DNA PCR 2 C 2 step PCR 98 C 10 sec. 68 C 5 10 5 sec. 10 DNA 108 cfu/ g pUC118 PCR 4 l 35 40 - PCR PCR 50 l 10 100 pg VIII PrimeSTAR Max dna polymerase R045A/B E.
9 Coli HST08 Premium Competent Cells 9128 E. coli JM109 Competent Cells 9052 E. coli DH5 Competent Cells 9057 E. coli HST02 Competent Cells 9127 IX PrimeSTAR 13 R046Av201601Da
