T4 DNA Ligase
Detailní popis
For in vitro use only!
Unit Definition: One Weiss unit is defined as the amount of enzyme required to catalyze the exchange of 1 nmol of 32P from pyrophosphate to ATP, into Norit-adsorbable material in 20 minutes at 37 °C.
Shipping: shipped on blue ice
Storage Conditions: store at -20 °C
avoid freeze/thaw cycles
Shelf Life: 12 months
Form: liquid (Supplied in 10 mM Tris-HCl pH 7.4, 50 mM KCl, 0.1 mM EDTA, 1 mM DTT, 200 μg/ml BSA and 50 % [v/v] glycerol)
Concentration: 2.5 Weiss units/μl (500 CE units/μl)
Description:
T4 DNA Ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5' phosphate and 3'-hydroxyl termini in duplex DNA or RNA.
Content:
Standard Ligation Buffer, 10x conc.
500 mM Tris-HCl pH 7.8 at 25 °C, 100 mM MgCl2, 100 mM DTT, 10 mM ATP and 25 μg/ml BSA
Fast Ligation Buffer, 2x conc.
60 mM Tris-HCl pH 7.8 at 25 °C, 20 mM MgCl2, 20 mM DTT, 2 mM ATP and 10 % PEG
| component | EN-149S | EN-149L |
| T4 DNA Ligase | 160 μl | 5 x 160 μl |
| Standard Ligation Buffer, 10x conc. | 1 ml | 5 x 1 ml |
| Fast Ligation Buffer, 2x conc. | 5 ml | 5 x 5 ml |
Heat inactivation:
T4 DNA Ligase can be inactivated by incubation at 65 °C for 10 minutes.
Note:
- One Cohesive-End Ligation Unit (CEU) is defined as the amount of enzyme required to give 50 % ligation of Hind III fragments of λ DNA (5' DNA termini concentration of 0.12 μM, 300 μg/ml) in a total reaction volume of 20 μl in 30 minutes at 16 °C in 1x T4 DNA Ligase Reaction Buffer.
- One Weiss unit is equivalent to approx. 200 CE units.
- T4 DNA Ligase is strongly inhibited by NaCl or KCl if the concentration exceeds 200 mM.
- Ligation of blunt-ended and single-base pair overhang fragments requires about 50 times as much enzyme to achieve the same extent of ligation as cohesive-end DNA fragments. Blunt-end ligation may be enhanced by addition of PEG 4000 (10 % w/v final concentration) or hexamine chloride, or by reducing the ATP concentration to 50 μM.
- To dilute T4 DNA Ligase for subsequent storage at -20 °C a storage buffer containing 50 % glycerol should be used, to dilute Ligase for immediate use, 1x Reaction Buffer is recommended.
Assay Set-Up:
Standard Ligation Assay
| comp. | final amount/conc. | 20 μl assay |
| Standard Ligation Buffer, 10x conc. | 1x | 2 μl |
| Vector/Insert DNA | 100 ng - 1 μg | 100 ng - 1 μg |
| T4 DNA Ligase | 0.1 - 1 Weiss units | 0.04-0.4 μl |
| PCR-grade Water | - | fill up to 20 μl |
Incubate for 20 - 30 min at 16 °C for optimal ligation.
Fast Ligation Assay
| comp. | final amount/conc. | 20 μl assay |
| Fast Ligation Buffer, 2x conc. | 1x | 10 μl |
| Vector/Insert DNA | 100 ng - 1 μg | 100 ng - 1 μg |
| T4 DNA Ligase | 0.1 - 1 Weiss units | 0.04-0.4 μl |
| PCR-grade Water | - | fill up to 20 μl |
Incubate for 5 min for cohesive-ended ligations or 15 min for blunt-ended ligations at ambient temperature (20 - 25 °C).
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