商品编号


M0369S-50 reactions



品牌


纽英伦(NEB)



公司


New England Biolabs



公司分类


DNA Modifying Enzymes




50 reactions

商品信息

Description:
ElectroLigase? combines T4 DNA ligase and an optimized, ready-to-use 2X reaction buffer containing a proprietary ligation enhancer and no PEG. This combination is specifically formulated to promote robust ligation of all types of DNA ends (blunt, sticky, TA). It is directly compat
IBL
e, without desalting or purification, with electrocompetent cells used for transformation by electroporation. No thawing of the buffer is required as it maintains a liquid state during storage at -20°C*, thereby simplifying reaction set-up. By providing an optimized ratio of enzyme and buffer components, users are able to rapidly ligate all types of DNA ends applying a short incubation time at room temperature. Ligations for subcloning can be carried out in small volumes with low concentrations, allowing users to conserve precious DNA samples. These reactions can be
Pipette
d directly, without purification or dilution, to transform many strains of electrocompetent
E. coli
**.
* Freezers vary in their actual internal temperatures. Our testing demonstrates that the enzyme and buffer remain liquid at -20°C.
**ElectroLigase is also compat
IBL
e with chemically competent strains of
E. coli
. Performance is generally around 50% efficiency, when compared to the Blunt/TA Ligase Master Mix (
NEB
#M0367 ).
Ligation reactions containing equal amounts (20 ng vectorand 3-fold molar excess of insert) of blunt- (A), T/A (B), or sticky-end (C)vector/insert pairs were set up using ElectroLigase and incubated for the timesshown. After heat inactivation, 2 μl of each reaction were withdrawn anddirectly used to transform
NEB
10-beta Electrocompetent
E. coli
(
NEB
#C3020). 50 μl aliquots of the outgrowth(diluted, in some cases) were plated onto selective plates and incubatedovernight at 37°C. Colonies were counted, adjusted for plating dilution, andgraphed.
Product Source
Purified from an
E. coli
strain containing a recombinant gene encoding T4 DNA Ligase.
Reaction Volume Definition
1X ElectroLigase Reaction Buffer with DNA substrates and 1 μl ElectroLigase in an 11 μl reaction volume incubated at 25°C for 30 minutes.
Notes:
Usage :Cells: Competent cells can vary by several logs in their competence. Perceived ligation efficiency directly correlates with the competence of the cells used for transformation. Always transform uncut vector as a control for comparison purposes.DNA: Purified DNA for ligations can be dissolved in dH2O (Milli-Q? water or equivalent is preferable); TE or other dilute buffers also work well. For optimum ligation, the amount of vector DNA should be 20–100 ng and the insert should be added at a 3-fold molar excess. For ligation volumes greater than 11 μl, increase the volume of ElectroLigase Reaction Buffer accordingly. Insert:vector ratios between 2 and 6 are optimal for single insertions. Ratios below 2:1 result in lower ligation efficiency. Ratios above 6:1 promote multiple inserts. If you are unsure of your DNA concentrations, perform multiple ligations with varying ratios.Time and Temperature: Most ligations performed using ElectroLigase reach an end point at 60 minutes or less when performed between 4–37°C. Incubation beyond this time provides no additional benefit. Our recommendation for a 25°C (room temperature) incubation was chosen after evaluation of performance at 4°C, 16°C, 25°C, and 37°C. Most conditions reached at least 50% performance within 30 minutes.
BIOLOG
y: Some DNA sequences are not easy to clone. Sequences that form structures, including inverted and tandem repeats, are selected against by E. coli. Some recombinant proteins are not well tolerated by E. coli and can result in poor transformation or small colonies