T4 DNA Ligase Buffer
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T4 DNA Ligase Buffer
Invitrogen™

T4 DNA Ligase Buffer

T4 DNA Ligase는 double-stranded DNA와 3´ hydroxyl 및 5´ phosphate termini 사이에 있는 ATP에서 phosphodiester 결합 형성을 촉매합니다. 고유한 T4자세히 알아보기
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카탈로그 번호수량
463000182 x 1 mL
카탈로그 번호 46300018
제품 가격(KRW)
80,000
Online offer
Ends: 31-Dec-2025
94,000
할인액 14,000 (15%)
Each
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수량:
2 x 1 mL
대량 주문 또는 맞춤형 요청
제품 가격(KRW)
80,000
Online offer
Ends: 31-Dec-2025
94,000
할인액 14,000 (15%)
Each
카트에 추가하기
T4 DNA Ligase는 double-stranded DNA와 3´ hydroxyl 및 5´ phosphate termini 사이에 있는 ATP에서 phosphodiester 결합 형성을 촉매합니다. 고유한 T4 DNA Ligase buffer가 5분 내에 ligation을 최적화합니다(1). Single-stranded nucleic acid는 이 효소의 기질로 사용되지 않습니다. T4 DNA Ligase Technical Bulletin을 이용할 수 있습니다.

어플리케이션: Cloning (blunt-end 또는 cohesive-end ligation) (2). Linker 또는 adapter를 blunt-ended DNA에 추가 (2).

기원: E. coli œ lysogen NM989에서 정제함.

성능 및 품질 검사: Endodeoxyribonuclease, 3´ 및 5´ exodeoxyribonuclease assays; ligation efficiency 검사 완료.

단위 정의: 1 단위가 37°C에서 20분 내에 1 nmol 32P-labeled pyrophosphate을 ATP로 가수분해합니다. (1 단위는 cohesive-end ligation unit 약 300개에 해당합니다.)

단위 반응 조건: 66 mM Tris-HCl (pH 7.6), 6.6 mM MgCl2 , 10 mM DTT, 66 μM ATP, 3.3 μM 32 P-labeled pyrophosphate, enzyme 0.1 ml - 20분, 37°C.
For Research Use Only. Not for use in diagnostic procedures.
사양
함께 사용가능한 버퍼5X Reaction Buffer
제품 유형T4 DNA Ligase Buffer
수량2 x 1 mL
배송 조건Approved for shipment on Wet or Dry Ice
Unit SizeEach
구성 및 보관
T4 DNA Ligase is supplied with a vial of 5X reaction buffer [250 mM Tris-HCl (pH 7.6), 50 mM MgCl2 , 5 mM ATP, 5 mM DTT, 25% (w/v) polyethylene glycol-8000]. Store at -20°C.

자주 묻는 질문(FAQ)

What are common inhibitors of the T4 DNA ligase?

dATP is a competitive inhibitor. Phosphate will reduce ligation efficiency. Detergents in your ligation buffer will likely not affect activity. High levels (0.2M) Na2+, K+, Cs+, Li+, and NH4+ inhibit the enzyme almost completely. Polyamines, spermine, and spermidine also serve as inhibitors.

Do both my insert and vector have to be phosphorylated for successful ligation?

At least one molecule in a ligase reaction (i.e., insert or vector) must be phosphorylated. Ligation reactions are dependent on the presence of a 5' phosphate on the DNA molecules. The ligation of a dephosphorylated vector with an insert generated from a restriction enzyme digest (phosphorylated) is most routinely performed. Although only one strand of the DNA ligates at a junction point, the molecule can form a stable circle, providing that the insert is large enough for hybridization to maintain the molecule in a circular form.

Which T4 DNA Ligase protocol do you recommend when ligating an insert containing one cohesive (sticky) end and one blunt end?

For cloning an insert with one cohesive end and one blunt end, use the conditions for blunt ends.  The sticky end may ligate quickly, but the blunt end ligation will still be inefficient. You should use the more stringent protocol to optimize the blunt end ligation. This usually means using more enzyme (5 U), a lower reaction temperature (14C) and a longer incubation time (16-24 hours). 

Why is it necessary to dilute ligated DNA products before adding them to competent bacterial cells?

Components of the ligation reaction (enzymes, salts) can interfere with transformation, and may reduce the number of recombinant colonies or plaques. We recommend a five-fold dilution of the ligation mix, and adding not more than 1/10 of the diluted volume to the cells. For best results, the volume added should also not exceed 10% of the volume of the competent cells that you are using.

What are the recommended conditions for blunt-ended ligations?

Generally, ligations are done in a 20 µL volume. Use a total of 100 to 1000 ng of DNA with an insert to vector ratio of 3:1. Add 1.0 units (Weiss) ligase to the reaction. Incubate at room temperature for 4 h or overnight at 14-16 degrees C.

Ideally, assemble several reactions with varying ratios of vector:insert (i.e. 3:1, 5:1, 10:1, 20:1, etc.) to determine the optimal ratio for ligation.

Thermo Fisher Scientific offers T4 DNA ligase at two concentrations: 1 U/µL (Cat. No. 15224-017) and 5 U/µL (Cat. No. 15224-041). When performing blunt or TA cloning ligations, the higher concentration of ligase is generally preferred since ligating a blunt or single base overhang requires more enzyme.

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