[ ]...1 [ ] Inverse PCR...2 [ ]...3 [ ]...4 Plasmid PCR Primer...4 PCR...5 PCR 2nd-site mutation...5 Plasmid...6 [ ]...8 Inverse PCR Dpn I
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- ひろみ みしま
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1 06-12 KOD - Plus- Mutagenesis Kit (Code No. SMK-101) TOYOBO CO., LTD. Life Science Department OSAKA JAPAN 0
2 [ ]...1 [ ] Inverse PCR...2 [ ]...3 [ ]...4 Plasmid PCR Primer...4 PCR...5 PCR 2nd-site mutation...5 Plasmid...6 [ ]...8 Inverse PCR Dpn I Plasmid...9 PCR Self-ligation [ ]...11 [ ] NOTICE TO PURCHASER: LIMITED LICENSE Use of this product is covered by one or more of the following US patents and corresponding patent claims outside the US: 5,079,352, 5,789,224, 5,618,711, 6,127,155 and claims outside the US corresponding to US Patent No. 4,889,818. The purchase of this product includes a limited, non-transferable immunity from suit under the foregoing patent claims for using only this amount of product for the purchaser s own internal research. No right under any other patent claim (such as the patented 5 Nuclease Process claims in US Patents Nos. 5,210,015 and 5,487,972), no right to perform any patented method, and no right to perform commercial services of any kind, including without limitation reporting the results of purchaser's activities for a fee or other commercial consideration, is conveyed expressly, by implication, or by estoppel. This product is for research use only. Diagnostic uses under Roche patents require a separate license from Roche. Further information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA. 1
3 PCR KOD -Plus- Inverse PCR ipcr Inverse PCR DNA 2 PCR PCR PCR PCR Self-ligation 1. Inverse PCR bp 10bp Tag 100bp 20 Mutant Clone Collection Saturation Mutagenesis KOD -Plus- PCR PCR 2nd-site mutation 10kbp 3. * PCR Self-ligation KinaseLigase PCR Primer 3 * 1
4 Inverse PCR Template Inverse PCR 510 A 0.52hr Template Dpn I plasmid B 371hr PCR Self-ligation C Kinase / Ligase 161hr Mutant D Transform E.coli 2
5 -20 No. 1 KOD -Plus- 1U/l (20 1 ) 25l 2 10x Buffer for ipcr 125l 3 2mM dntps 125l 4 Dpn I(10U/l) 50l 5 T4 Polynucleotide Kinase(5U/l 50l 6 Ligation high 250l 7 Control Plasmid pak119m (50ng/l) 10l 8 Control Primer #1 (10 pmol/l) 10l 9 Control Primer #2 (10 pmol/l) 10l 1) 20 5 LB 50mg/ml 20mg/ml 4% X-gal 100mMIPTG Plasmid Blue/White Competent high DH5 Code No.:DNA-903 JM109 Code No.:DNA-900 3
6 Plasmid PCR Plasmid Dpn I Dpn G m6 ATC DNA Plasmid DNA JM109DH5 Plasmid dam methylase dam JM110 SCS110 Plasmid Plasmid in vitro dam methylase JM109 dam Plasmid PCR Primer PCR bp 25bp PCR PCR KOD -Plus- 3'5' Exonuclease Proofreading 3 Inverse PCR PCR 5 HPLC 5 HPLC 40mer PAGE 4
7 Substitution Deletion Insertion or Template plasmid PCR Inverse PCR PCR ipcr buffer PCR Plasmid PCR1020 PCR PCR PCR 2nd-site mutation PCR KOD Plus 2nd-site mutation 2nd-site mutation 5
8 Plasmid Plasmid lacz Plasmid(pAK119M) lacz 6 TAA stop Plasmid X-gal TAA stop CCA Pro 80 Plasmid 6
9 pak119m 4.3 kb lacz TAAstop White Colony CCAPro Blue Colony LacZ Plasmid 6 7
10 Inverse PCR PCR 10 pmol/l Plasmid DNA 50ng/l PCR 35l 10Buffer for ipcr 5l 2 mm dntps 5l 1 10 pmol/l 1.5l 2 10 pmol/l 1.5l PlasmidDNA 50ng/l 1l KOD -Plus- 1l Total Volume 50l PCR 94 2 min sec 68 X min 4 Hold 1 2 Y kb 1min./kb 5 Plasmid 5min 2) kb 1 /kb PCR l 10Buffer for ipcr 5l 2 mm dntps 5l Control Primer #1 10 pmol/l 1.5l Control Primer #2 10 pmol/l 1.5l Control Plasmid pak119m 50ng/l 1l KOD -Plus- 1l Total Volume 50l 94 2min 9810sec 68 5min 4 Hold 5 8
11 Dpn I Plasmid PCR 50l Dpn I 2l ) PCR 2 PCR Dpn I 1l 37 ) PCR Dpn I 5l PCR 5 PCR Self-ligation T4 Polynucleotide Kinase Ligation high Ligation high PCR Dpn I PCR Ligation high T4 Polynucleotide Kinase Total Volume 2l 7l 5l 1l 15l 16 1 ) 9
12 Competent high DH5 Code No. DNA 903 Competent high JM109 Code No. DNA 900 Competent Cell Self- igation pbr322 pbr3221pg 100 l 1x10 9 cfu/µg-pbr SOC LB 10200l LB/ /IPTG/X-gal LB/ /IPTG/X-gal hr 1x10 9 cfu/µg-pbr l Plasmid Plasmid PCR 10
13 Inverse PCR PCR Inverse PCR 10 8 cfu/µg-pbr322 Plasmid In vitro dam methylase dam Plasmid [4](1) 4 1 Inverse PCR PCR PCR Plasmid Inverse PCR PCR Plasmid Plasmid In vitro dam methylase dam Plasmid [4](1) IPTGX-gal LB/ /IPTG/X-gal / IPTGX-gal DH5 JM109 11
14 LB (1L) 10g bacto-tryptone 5g bacto-yeast extract 10g NaC 1L NaOH ph7.2 LB/ 1LLB 15g g/ml LB/ LLB 15g 50 20g/ml 100mMIPTG (10mL) 0.24g IPTG 10ml -20 4% X-gal (10mL) 0.4g X-gal 5-bromo-4-chloro-3-indolyl--D-galactoside N,N-dimethyl- formamide DMF 10ml -20 LB/ /IPTG/X-gal LB/ 100mM IPTG4% X-gal 20l 12
15 Code No. KOD -Plus- < PCR > 200U KOD-201 Dpn I 1,000U DPN-101 T4 Polynucleotide Kinase 1,500U 1 PNK-111 1,500U 5 PNK-112 Ligation high< > 50 LGK-101 MagExtractor -Plasmid- < Plasmid DNA > MagExtractor -PCR & Gel Clean up- < DNA fragment > 500 NPK NPK-601 Magical Trapper< > MGS-101 Competent high DH5 < E. DH5 SOC > Competent high JM109 < E. JM109 SOC > 100l 10 DNA l 10 DNA
16 TEL FAX order_lifescience@bio.toyobo.co.jp TEL FAX order_lifescience@bio.toyobo.co.jp TEL FAX :0012:00, 13:0017:00 techosk@bio.toyobo.co.jp URLhttp:// 14
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