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
B1010 (2X Rapid Ligation Buffer)
B6030 (10X T4 DNA Ligase Buffer)
2X Rapid Ligation Buffer (B1010):
132mM Tris-HCI
20 mM MgCl2
2 mM DTT
2 mM ATP
15% PEG 6000
pH 7.6 @ 25°C
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 DNALigase required to join 50% of 100 ng of DNA fragments with cohesive termini in50 μl 1X DNA Ligase Buffer following a 30 minute incubation at 23°C.
PRODUCT INFORMATION产品信息
T4 DNA Ligase (Rapid)
Part Number L6030-HC-L
Concentration 600,000 U/ml
Unit Size 240,000 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 = no conversion
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
10 μL 2X Rapid Ligation Buffer 1X
X μL Vector 1-10 ng/μL
X μL Insert 1-10 ng/μL
1 μL T4 DNA Ligase (600 U/ μL) 30 U/μL
X μL Type I Water N/A
20 μL Total Volume
Add all of above components to a cleanreaction vessel, mix well by pipetting.
Incubate at 25°C for 10 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-insertligations are optimal when targeting an insert:vector ratio between 2 and 6. Aratio above 6:1 will promote the insertion of multiple fragments, whiledropping below 2:1 will reduce ligation efficiency.
For problematic ligations or if the DNAconcentration is unknown, it may be necessary to vary ratios and run multipleligations.
The presence of PEG at a high concentrationwill significantly reduce the transformation efficiency of electrocompetentcells. In order to maximize the efficiency of transformation intoelectrocompetent cells, the following approaches are recommended:
Best: Following ligation, purify theproduct using a DNA purification spin column and elute in 50 μL of TE. The DNAis now ready for transformation. The final amount of DNA to be transformedshould be in the range of 0.1-10 ng.
Better: Dilute ligation product in ddH20or TE to reduce the PEG concentration. The final amount of DNA to betransformed should be in the range of 0.1-10 ng.
Enzymatics’ high-concentration T4 DNALigase in combination with the 2X Rapid Ligation buffer greatly stimulates therate and efficiency blunt-end ligation, therefore long incubations (>10minutes) are NOT recommended and can greatly reduce the transformationefficiency of ligation products. In order to maximize transformation efficiencyof the correct insert/vector combination, the following protocol is recommended.
Enzymatics 10X T4 DNA Ligase Buffer doesnot contain PEG and is compatible with standard ligation protocols which do notspecify the use of a rapid/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.
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