PRODUCT DESCRIPTION产品描述
T4 DNA Ligase catalyzes the formation of aphosphodiester bond between the terminal 5′ phosphate and a 3′hydroxyl groups of duplex DNA or RNA. The enzyme efficiently joinsblunt and cohesive ends and repairs single stranded nicks in duplex DNA, RNA orDNA/RNA hybrids.
Source of Protein蛋白来源
A recombinant E. coli strain carrying thecloned T4 DNA Ligase gene.
Supplied In
10 mM Tris-HCl
50 mM KCl
1 mM DTT
0.1 mM EDTA
50% glycerol
pH 7.5 @ 25°C
Supplied With
B6030 (10X T4 DNA Ligase Buffer)
10X T4 DNA Ligase Buffer (B6030)
500mM Tris-HCI
100 mM MgCl2
50 mM DTT
10 mM ATP
pH 7.6 @ 25°C
Unit Definition单位定义
1 unit is defined as the amount of T4 DNALigase required to join 50% of 100 ng of DNA fragments with cohesive termini in50 μl 1X T4 DNA Ligase Buffer following a 30 minute incubation at 23°C.
PRODUCT INFORMATION产品信息
T4 DNA Ligase
Part Number L6030-LC-L
Concentration 120,000 U/ml
Unit Size 150,000 U
SDS Available on request
PRODUCT SPECIFICATION*产品特性
Storage Temperature -25 to -15°C
Test Specification
Purity (SDS-PAGE) >99%
Specific Activity 300,000 U/mg
SS Exonuclease 6,000 U <1.0%released
DS Exonuclease 6,000 U <1.0%released
DS Endonuclease 6,000 U = noconversion
E.coli DNA Contamination 6,000 U <10 copies
* For a detailed summary of assayconditions and data, refer to the Quality Controls Analysis section.
USAGE INSTRUCTIONS使用说明
Reaction Set-Up:
Amount Description Final Concentration
2 μL 10X T4 DNA Ligation Buffer 1X
X μL Vector 1-10 ng/μL
X μL Insert 1-10 ng/μL
1 μL T4 DNA Ligase (120 U/ μL) 6 U/μL
X μL TypeI Water N/A
20 μL TotalVolume
Addall of above components to a clean reaction vessel, mix well by pipetting.
Incubate at 25°C for 30 minutes.
Immediately purify DNA using PCR clean-upcolumn and elute in ~50 μL.
- OR–Immediately dilute (at least 1:10, but enough such that 0.1-10 ng ofligation product will be transformed) in TE or water
Transform 0.1-10 ng of ligation productinto chemically or electrocompetent cell line that is compatible with vector
NOTES
One Enzymatics T4 DNA Ligase cohesive endunit is equivalent to approximately 3 cohesive end units as measured with aLambda-Hind III DNA fragment substrate in 1X T4 DNA Ligase reaction buffer.
One Weiss Unit is approximately equivalentto 22 Enzymatics cohesive end units.
T4 DNA Ligase is ATP dependent. It isrecommended that the reaction buffer be discarded after one year of storage at-20°C andreplaced with fresh buffer to ensure maximum performance.
Single-insert ligations are optimal whentargeting an insert:vector ratio between 2 and 6. A ratio above 6:1 willpromote the insertion of multiple fragments, while dropping below 2:1 willreduce ligation efficiency. For problematic ligations or if the DNAconcentration is unknown, it may be necessary to vary ratios and run multipleligations. Enzymatics 10X T4 DNA Ligase Buffer does not contain PEG and iscompatible with standard ligation protocols which do not specify the use of arapid/fast/quick format buffer.
REFERENCES参考文献
Engler, M.J. and Richardson, C.C. (1982)P.D. Boyer (Eds.), The Enzymes, 5, pp. 3. San Diego: Academic Press.
Limitations of Use适用范围
This product was developed, manufactured,and sold for in vitro use only. The product is not suitable for administrationto humans or animals.
www.hyqbio.com.cn