T4 DNA Ligase (5 U / μL)

 

Khoom npe	Cat. Tsis muaj.	Spec.
T4 DNA Ligase (5 U / μL)	G3340-50	250 U
	G3340-100	500 U

 

 

T4 DNA Ligase catalyzes tsim cov phosphodiester daim ntawv cog lus ntawm juxtaposed 5 'phosphate thiab 3' hydroxyl termini nyob rau hauv duplex DNA los yog RNA. Cov enzyme no yuav koom nrog blunt kawg thiab cohesive kawg termini nrog rau kho ib leeg stranded nicks hauv duplex DNA thiab qee qhov DNA / RNA hybrids.

Lub ntsiab lus ntawm Kev Ua Haujlwm: Ib chav Weiss ntawm cov enzyme catalyzes hloov pauv ntawm 1 nmol ntawm [32PPi] rau hauv ATP hauv 20 min ntawm 37 degree. Ib chav Weiss yog sib npaug rau kwv yees li 200 cohesive kawg ligation units (CEU).

T4 DNA Ligase Storage Buffer: 20 mM Tris-HCl (pH 7.5), 50 mM KCl, 1 mM DTT, 50% (v/v) glycerol.

5 × T4 DNA Ligase Buffer: 250 mM Tris-HCl (pH 7.6), 50 mM MgCl2, 5 mM ATP, 5 mM DTT, Enhancer.

 

 

Nkoj nrog dej khov; khaws cia ntawm -20 degree, siv tau rau 12 lub hlis.

 

 

Tus naj npawb	Cheebtsam	G3340-50	G3340-100
G3340-1	T4 DNA Ligase	250 U (50 μL)	500 U (2×50 μL )
G3340-2	5 × T4 DNA Ligase Buffer	1 ml ib	2 x 1ml
Phau ntawv		Ib daim ntawv luam

 

 

1. Ntxiv cov hauv qab no (pom zoo 10-uL cov tshuaj tiv thaiv kab mob) rau ib qho autoclaved, 1.5-mL microcentrifuge raj:

Cheebtsam	Ntim
5 × T4 DNA Ligase Buffer	2 μL
T4 DNA Ligase	0.5-1 μL
Linear vector DNA	X μL
Ntxig DNA	Y μL
Dej tsis muaj Nuclease	Rau 10μL
Tag nrho	10 μL

2. Sib tov maj mam thiab centrifuge luv luv kom coj cov ntsiab lus mus rau hauv qab ntawm lub raj.

3. Rau qhov sib xyaw ua ke, ua rau ntawm 25 degree rau 5-30 feeb; Rau blunt kawg, incubate ntawm 25 degree tsawg tshaj li 2 teev los yog hmo ntuj ntawm 4 degree.

4. Muab lub raj tso rau ntawm cov dej khov thiab ua tam sim ntawd los ua cov tshuaj tiv thaiv kev hloov pauv. Lossis koj tuaj yeem khaws cov sib tov sib xyaw ntawm -20 degree kom txog thaum koj npaj tau.

Ua cov tshuaj tiv thaiv kev hloov pauv

5. Ntxiv cov sib tov tsim nyog rau hauv cov tshuaj muaj peev xwm (xws li E.coli DH5 , E.coli Top10, thiab lwm yam) thiab sib tov los ntawm tapping maj mam. Tsis txhob sib tov los ntawm pipetting nce thiab nqis. Qhov sib xyaw ligation ntxiv tuaj yeem khaws cia ntawm -20 degree .

6. Muab tso rau hauv dej khov rau 30 feeb.

7. Incubate rau raws nraim 90 vib nas this hauv 42 degree da dej. Tsis txhob sib tov lossis co.

8. Tshem cov raj centrifuge los ntawm 42 degree da dej thiab muab tso rau hauv dej khov rau 2-5 feeb.

9. Ntxiv 900 µL ntawm SOC lossis LB nruab nrab. Cov txheej txheem sterile yuav tsum tau xyaum kom tsis txhob muaj kab mob. Co lub centrifuge raj ntawm 37 degree rau 1 teev ntawm 225 rpm nyob rau hauv ib tug co incubator.

10. Tshaj tawm cov ntim tsim nyog los ntawm txhua qhov hloov pauv centrifuge raj ntawm nyias, sau npe LB agar daim hlau. Cov kev hloov pauv ntxiv tuaj yeem khaws cia ntawm 4 degree thiab plated tawm hnub tom qab, yog tias xav tau.

11. Hloov lub phaj thiab ua rau ntawm 37 degree thaum hmo ntuj.

Txheeb xyuas qhov hloov pauv

12. Xaiv cov cheeb tsam thiab txheeb xyuas los ntawm kev sib cais ntawm plasmid, PCR, lossis kev sib txuas.

 

 

1. Nws raug pom zoo tias cov tshuaj tiv thaiv yuav tsum tau npaj rau ntawm cov dej khov.

2. Ib tug molar ratio ntawm 3: 1 ~ 10: 1 insert: vector yog pom zoo rau lub ligation ceev ntawm DNA inserts rau vectors los tsim ib ncig recombinant molecules.

3. Ua ntej siv, thaw 5X DNA Ligase Reaction Buffer nyob rau hauv chav tsev kub thiab vortex vigorously rau dissolve tej khoom precipitated.

4. T4 DNA Ligase yuav tsum tau khaws cia ntawm -20 degree kom txog thaum nyob rau hauv 5-10 feeb ntawm kev siv thiab rov qab tam sim ntawd rau -20 degree tom qab siv.

5. Yog hais tias ntxig DNA yog blunt kawg, lub vector nram qab no txwv endonuclease digestion yuav tsum dephosphorylated (pom zoo G3400) tiv thaiv nws tus kheej-circularization.

6. Rau koj txoj kev nyab xeeb thiab kev noj qab haus huv, thov hnav tsom iav, hnab looj tes, lossis khaub ncaws tiv thaiv.

 

 

Cim npe nrov: Tuam Tshoj t4 dna ligase (5 u/ul) manufacturers, lwm tus neeg, Hoobkas