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1、诺唯赞生物科技T4 DNA Ligase (Rapid)Catalog #N103IntroductionT4 DNA ligase catalyzes the formation of a phosphodiester bond between 5 terminal phosphate and adjacent 3 hydroxyl termini in duplex DNA, RNA or DNA/RNA hybrids. This enzyme will join blunt end and cohesive end termini but it cannot repair single
2、 stranded nicks.Package InformationComponentN103-01 600,000 UT4 DNA Ligase (Rapid) (600 U/此1 mL2 x Rapid Ligation Buffer6 mL10 x T4 Rapid Ligase Buffer2 mLReaction Buffer2 x Rapid Ligation Buffer132 mM Tris-HCI pH 7.6 2520 mM MgCl22 mM DTT2 mM ATP15% PEG 600010 x T4 Rapid Ligase Buffer500 mM Tris-HC
3、I pH 7.6 25 100 mM MgCh50 mM DTT10 mM ATPStorageStore at -20.Unit DefinitionOne unit is defined as the amount of enzyme required to give 50% ligation of Hindlll fragments of X DNA (100ng)in a total reaction volume of 50 |il in 30 minutes at 23 in lx T4 DNA Ligase Reaction Buffer.Quality ControlExonu
4、clease Activity: Incubation of 2000 U of this product and 0.6 |ig of X-Hind III at 37 for 16 hour results in nodetected change in DNA bands after gel electrophoretic.诺唯赞生物科技曾等公然戏5Vazyme Biotech CO.Ltd. 销售咨询:中国江苏省南京经济开发区科创路红枫科技园C1-2栋诺唯赞生物科技Endonuclease Activity: Incubation of 2000 U of this product a
5、nd 0.6 |ig of Supercoiled pBR322 DNA at 37 for 4 hour results in no detected change in DNA bands after gel electrophoretic.ProtocolApplication example 1 :Connect DNA and carrier1. Prepare the ligation reaction mixture in a microcentrifuge tube:Sterile distilled waterUp to 10 pl10x Ligase Buffer1 plI
6、nserta0.3 pmolVectorb0.3 pmolT4 DNA Ligase (Rapid) (600 U / pl)1 pla.The molar ratio of the insert and vector should be among 3:1 to 10:1.b.For vector with blunt terminal, please perform the dephosphorylation of vector to prevent cyclization.l.Incubate the reaction mixture at 16 overnight.3.T ransfo
7、rmationl)Take the competent cells out of the -80O C refrigerator, and place the competent cells immediately in an ice water bath.2)Add the DNA into 100 n 1 of competent cells and mix gently. Keep in the ice for 30 minutes.3)Incubate the mixture at 42 for 90 seconds. And then return to the ice bath f
8、or 23 minutes.4)Add SOC or LB medium up to a final volume of 1 mL keep in water bath at 37 for 10 minutes.5)Culture by shaking (150 rpm) at 37for 45 minutes to recovery.6)Centrifuged at 2500 g for 5 minutes, and then remove 900 H 1 of the supernatant. Mix the remaining solution and plate on selectiv
9、e media.7)Incubate at 37overnight.Application example 2:Adaptor ligation in the DNA library construction1. Prepare the ligation reaction mixture in a microcentrifuge tube:dA-Tailing Products10 pl2 x Rapid Ligation Buffer15 plDNA Adapter2.5 plT4 DNA Ligase (Rapid) (600 U / pl)2.5 plMix reaction by gently pipetting (DO NOT VORTEX) followed by a briefly centrifugation so the reaction volume collects at the bottom of the PCR tube.2.Place the reaction tube in the PCR instrument and operate under the following condition:30 10 min4Hold