貨號(hào):L6030-HC-L
產(chǎn)品描述:
T4 DNA連接酶催化雙鏈DNA或RNA的末端5'磷酸和3'羥基之間形成磷酸二酯鍵。該酶有效連接鈍端和粘端,并修復(fù)雙鏈DNA,RNA或DNA / RNA雜合體中的單鏈缺口(1)。
規(guī)格: | 600,000 U/ml |
濃度: | 240,000 U |
保存: | -25°C至-15°C |
純度(SDS-PAGE) | > 99% |
活性: | 300,000 U/mg |
SS核酸外切酶 | 6,000 U< 1.0 % Released |
DS核酸外切酶 | 6,000 U< 1.0 % Released |
DS內(nèi)切核酸酶 | 6,000 U=No Conversion |
大腸桿菌 DNA污染 | 6,000 U <10 copies |
蛋白質(zhì)來源
攜帶克隆的T4 DNA連接酶基因的重組大腸桿菌菌株。
提供:
10 mM Tris-HCl
50 mM KCl
1 mM DTT
0.1 mM EDTA
50% glycerol
pH 7.5 @ 25°C
隨附:
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
單位定義
1單位定義為在23℃孵育30分鐘后,在50μl1X DNA連接酶緩沖液中加入100ng具有粘性末端的DNA片段的50%所需的DNA連接酶的量。
使用說明:
1、反應(yīng)設(shè)置
Amount | Description | Final Concentration |
---|---|---|
10 uL | 2X Rapid Ligation Buffer | 1X |
X uL | Vector | 1-10 ng/uL |
X uL | Insert | 1-10 ng/uL |
1 uL | T4 DNA Ligase (600 U/ uL) | 30 U/uL |
X uL | Type I Water | N/A |
20 uL | Total Volume |
|
2、過程操作
1)將所有上述組分加入干凈的反應(yīng)容器中,通過移液充分混合。
2)25°C孵育10分鐘。
3)使用PCR清潔柱立即純化DNA并洗脫約50μL。
4)- 或 - 在TE或水中立即稀釋(至少1:10,但足以使0.1-10ng連接產(chǎn)物轉(zhuǎn)化)
5)將0.1-10ng連接產(chǎn)物轉(zhuǎn)化為與載體相容的化學(xué)或電子感受態(tài)細(xì)胞系
3、其他
在1X T4 DNA連接酶反應(yīng)緩沖液中用Lambda-Hind III DNA片段底物測量,一個(gè)酶酶T4 DNA連接酶粘性末端單位相當(dāng)于約3個(gè)粘性末端單位。
一個(gè)Weiss單位大約相當(dāng)于22個(gè)Enzymatics粘性末端單位。
T4 DNA連接酶是ATP依賴性的。建議在-20°C儲(chǔ)存一年后丟棄反應(yīng)緩沖液,并用新鮮緩沖液代替以確保*性能。當(dāng)靶向插入片段:載體比率在2和6之間時(shí),單插入連接是*的。高于6:1的比率將促進(jìn)插入多個(gè)片段,而降至2:1以下將降低連接效率。
對于有問題的連接或DNA濃度未知的情況,可能需要改變比率并進(jìn)行多次連接。
高濃度PEG的存在將顯著降低電感受態(tài)細(xì)胞的轉(zhuǎn)化效率。為了zui大限度地轉(zhuǎn)化為電感受態(tài)細(xì)胞,建議采用以下方法:
*: 連接后,使用DNA純化離心柱純化產(chǎn)物并在50μLTE中洗脫。DNA現(xiàn)在已準(zhǔn)備好轉(zhuǎn)化。要轉(zhuǎn)化的DNA的zui終數(shù)量應(yīng)該在0.1-10ng范圍內(nèi)。
更好: 在ddH2O或TE中稀釋連接產(chǎn)物以降低PEG濃度。要轉(zhuǎn)化的DNA的zui終數(shù)量應(yīng)該在0.1-10ng范圍內(nèi)。
Enzymatics的高濃度T4 DNA連接酶與2X快速連接緩沖液結(jié)合使用,極大地刺激了平端連接的速度和效率,因此不建議長時(shí)間孵育(> 10分鐘),并可能大大降低連接產(chǎn)物的轉(zhuǎn)化效率。為了使正確的插入/載體組合的轉(zhuǎn)化效率zui大化,推薦使用以下方案。
酶學(xué)10X T4 DNA連接酶緩沖液不含PEG,并且與標(biāo)準(zhǔn)連接方案相兼容,該方案不規(guī)定使用快速/快速/快速格式緩沖液。
部分產(chǎn)品信息:
貨號(hào) | 品名 | 規(guī)格 | 濃度 | 品牌 |
---|---|---|---|---|
N2070-10-L | ATP Solution | 50 µmol | 10 mM | Enzymatics |
N2050-10-L | 10 mM dNTP Solution Mix | 100 µmol | 10 mM | Enzymatics |
N2010L | 100 mM 75µmol dNTP Solution Set | 75 µmol | 100 mM | Enzymatics |
N2010-A-L | 100 mM dATP Solution | 75 µmol | 100 mM | Enzymatics |
N2050L | 100µmol dNTP Solution Mix | 100 µmol | 25 mM | Enzymatics |
Y9180L | 10x Uracil Cleavage System | 750 Reactions | N/A | Enzymatics |
P7670 | 2x HiFi PCR Master Mix | 1 rxn | 2x | Enzymatics |
Y9420L | 5x ER/A-Tailing Enzyme Mix | 24 reactions | N/A | Enzymatics |
Y9380L | E. coli Pyrophosphatase | 50 U | 100 U/mL | Enzymatics |
Y9030L | E. coli Single-Stranded DNA Binding Protein | 1.5 mg | 2.75 mg/ml | Enzymatics |
Y9140-HC-L | End-Repair Mix (High Concentration) | 75 Reactions | 1 Rxn/µL | Enzymatics |
Y9140-LC-L | End-Repair Mix (Low Concentration) | 200 Reactions | 1 Rxn/µL | Enzymatics |
Y9080L | Endonuclease VIII | 10,000 U | 10,000 U/ml | Enzymatics |
P7600L | EnzScript™ (MMLV Reverse Transcriptase RNase H-) | 10,000 U | 200,000 U/mL | Enzymatics |
X8010L | Exonuclease I | 30,000 U | 20,000 U/ml | Enzymatics |
X8020L | Exonuclease III | 50,000 U | 100,000 U/ml | Enzymatics |
P7010-HC-L | Klenow (3′→ 5′ exo-) (High Concentration) | 10,000 U | 50,000 U/ml | Enzymatics |
P7010-LC-L | Klenow (3′→ 5′ exo-) (Low Concentration) | 10,000 U | 5,000 U/ml | Enzymatics |
P7060L | Klenow Fragment | 2,500 U | 5,000 U/ml | Enzymatics |
X8030L | Lambda Exonuclease | 10,000 U | 5,000 U/ml | Enzymatics |
P7040L | M-MuLV Reverse Transcriptase | 100,000 U | 200,000 U/ml | Enzymatics |
P7090L | Mako DNA Polymerase (3′→ 5′ exo-) | 3,000 U | 30,000 U/ml | Enzymatics |
P7140-HC-L | Manta 1.0 DNA Polymerase (High Concentration) | 100,000 U | 400,000 U/ml | Enzymatics |
P7140-LC-L | Manta 1.0 DNA Polymerase (Low Concentration) | 100,000 U | 40,000 U/ml | Enzymatics |
P7610L | NGS Library Prep: 2X VeraSeq™ PCR Mix – 250 Reactions | 250 Reactions | 2X | Enzymatics |
P7590L | Phoenix™ Hot Start Taq DNA Polymerase | 500 U | 5,000 U/ml | Enzymatics |
P7460L | Poly(A) Polymerase | 1,000 U | 5,000 U/mL | Enzymatics |
Y9260L | RecA | 1.5 mg | 2.0 mg/mL | Enzymatics |
Y9220L | RNAse H | 5,000 U | 5,000 U/ml | Enzymatics |
Y9240L | RNAse Inhibitor | 20,000 U | 40,000 U/ml | Enzymatics |
L6010L | T3 DNA Ligase | 900,000 U | 3,000,000 U/ml | Enzymatics |
L6030-LC-L | T4 DNA Ligase | 150,000 U | 120,000 U/ml | Enzymatics |
L6030-HC-L | T4 DNA Ligase (Rapid) | 240,000 U | 600,000 U/ml | Enzymatics |
P7080L | T4 DNA Polymerase | 2,000 U | 3,000 U/ml | Enzymatics |
Y9130L | T4 Gene 32 Protein | 1.0 mg | 10 mg/mL | Enzymatics |
Y9040L | T4 Polynucleotide Kinase | 10,000 U | 10,000 U/ml | Enzymatics |
L6050L | T4 RNA Ligase 1 | 10,000 U | 20,000 U/ml | Enzymatics |
L6080L | T4 RNA Ligase 2 | 4,500 U | 30,000 U/ml | Enzymatics |
L6070L | T4 RNA Ligase 2 Truncated | 500 U | 5,000 U/ml | Enzymatics |
L6020L | T7 DNA Ligase | 900,000 U | 3,000,000 U/ml | Enzymatics |
P7260L | T7 DNA Polymerase | 3,500 U | 10,000 U/ml | Enzymatics |
P7180L | T7 RNA Polymerase | 50,000 U | 50,000 U/ml | Enzymatics |
L6060L | Taq DNA Ligase | 20,000 U | 40,000 U/ml | Enzymatics |
P7110L | Taq DSC 2.0 DNA Polymerase | 1,250 U | 5,000 U/ml | Enzymatics |
P7250L | Taq-B DNA Polymerase | 10,000 U | 5,000 U/ml | Enzymatics |
P7620L | TaqIT | 5,000 U | 50,000 U/mL | Enzymatics |
P7070L | Terminal deoxynucleotidyl Transferase (TdT) | 6,000 U | 20,000 U/ml | Enzymatics |
G5020L | Thermolabile UDG | 500 U | 1,000 U/ml | Enzymatics |
Y9370L | Thermostable Pyrophosphatase | 1,250 U | 2,000 U/ml | Enzymatics |
G5010L | Uracil DNA Glycosylase | 10,000 U | 2,000 U/ml | Enzymatics |
P7511L | VeraSeq™ 2.0 High-Fidelity DNA Polymerase | 500 U | 2,000 U/ml | Enzymatics |
P7520L | VeraSeq™ ULtra DNA Polymerase | 500 U | 2,000 U/ml | Enzymatics |
L6030-W-L | WGS Ligase | 24 Reactions | N/A | Enzymatics |
P7640L | ZipScript™ One-Step RT-qPCR Kit | 1000 Reactions | N/A | Enzymatics |
P7020-HC-L | φ29 DNA Polymerase (High Concentration) | 2,000 U | 100,000 U/ml | Enzymatics |
P7020-LC-L | φ29 DNA Polymerase (Low Concentration) | 2,000 U | 10,000 U/ml | Enzymatics |
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酶產(chǎn)品在生命科學(xué)研究中是*的重要工具,在科學(xué)研究中對于其性能、純度和穩(wěn)定性的要求日益提升?;蚬?代理的酶產(chǎn)品專家—Enzymatics一貫為客戶提供zui高質(zhì)量和*價(jià)值的產(chǎn)品,因此我們竭誠向您推薦 Enzymatics產(chǎn)品。
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