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DESCRIPTION Features and applications
T4 DNA Ligase catalyzes the joining of two strands of DNA between the 5'-phosphate and the 3'-hydroxyl groups of adjacent nucleotides in either a blunt-ended or cohesive-ended configuration. T4 DNA Ligase catalyzes the joining of RNA to either DNA or RNA strands in a duplex molecule but will not join single-stranded nucleic acids. T4 DNA Ligase is ATP dependent. Recommendations It is recommended that the ATP solution provided is aliquoted into smaller vials that can be defrosted when required. Repeated freeze/thawing of this solution is not recommended.
PCR Reaction Conditions (for a 20 μl volume)
Run the DNA ligation for 1-2 hours at room temperature at 20 - 25° C or 12-16 hours at 12° C. The total DNA concentration should be no more than 100 ng. The recommended Insert : Vektor ratio should be between 6 and 2. A smaller ratio will result in a less efficient ligation, whilst a higher ratio will incite multiple insertions. This data is intended for use as a guide only; conditions will vary from reaction to reaction and may need optimization.
Reagent Specifications 10x Reaction Buffer: 660 mM Tris-HCl (pH 7.6), 50 mM MgCl, 100 mM DTT. Storage and Dilution Buffer 10 mM Tris-HCl, pH 7.4, 100 mM NaCl, 1 mM DTT, 0.1 mM EDTA and 50 % glycerol. Storage Conditions T4 DNA Ligase can be stored at -20° C in a constant temperature freezer for 12 months. Q/C Assay Conditions Complete ligation of cohesive-ended Hind III-fragments was achieved using only 0.01 units of enzyme per mg of Lambda DNA when incubated for 30 minutes at 16° C. Efficient ligation of blunt-ended Hae III fragments was achieved using 1.0 units of enzyme per mg of DNA when incubated for 30 minutes at 16° C. Unit Definition One Weiss unit is defined as the amount of enzyme required to catalyze the conversion of 1nmole of 32P pyrophosphate into Norit-adsorbable material in 66mM Tris HCl (pH 7.6), 6mM MgCl2, 1mM DTT, 0.066mM ATP and 3.3 mM [32PPi] at 37° C for 20 minutes (2). 0.014 Weiss unit of ligase is equivalent to 1 ligation unit which catalyzes greater than 95 % ligation of 1mg of Lambda/Hind III fragments at 16° C in 20 minutes.
DESCRIPTION Features and applications
PAN Ligation is designed to carry out fast and efficient ligation of both cohesive and blunt ended DNA at room temperature. PAN Ligation will ligate 99 % of λ/HindIII cohesive-ended fragments, or 80 % of λ/EcoRV blunt-ended fragments, Storage Conditions PAN Ligation Kit can be stored for 12 months at -20° C. Avoid multiple freeze/thaw cycles. Storage and Dilution Buffer 10mM Tris-HCl, pH 7.4, 100mM NaCl, 1 mM DTT, 0.1mM EDTA and 50 % glycerol. Q/C Assay Conditions Each batch of PAN Ligation is tested for ligation to a single band, of the products of both HindIII and EcoRV cut Lambda DNA. The ligated DNA is then re-cut to ensure no alteration of restriction pattern.
DESCRIPTION Features and applications
Proteinase K is an enzyme used to digest most proteins in molecular-biological techniques. The enzyme may be used at 56° C for up to 4 hours, or 37° C for overnight incubations. Proteinase K solution is stabilized with a specially formulated buffer, and can be used directly from the freezer. Recommendations for Use - Dissolve to 20 mg/ml in 50 mM Tris-HCl, 2 mM calcium acetate, pH 8.0 Storage Conditions Proteinase K can be stored for 12 months at -20° C. Contaminants RNase Activity: No detectable ribonuclease activity detected with MS2RNA after 6-hour incubation at 37° C Unit definition One unit is defined as the amount of enzyme that will liberate 1.0 μmol of tyrosine per minute at 37° C, pH 7.5.
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