What is fast ap?

FastAP is a novel alkaline phosphatase, which is active in all Thermo Scientific restriction enzyme buffers as well as in PCR buffers. It dephosphorylates all types of DNA ends (blunt, 5′- and 3′-overhangs) in 10 minutes at 37°C.

How do you turn off alkaline phosphatase?

Inactivation. Alkaline phosphatase can be inactivated by heat: Heat the reaction for 10 min at +65 °C followed by at least one extraction with phenol/chloroform/isoamylalcohol (50 : 48 : 2). AP inactivation by heat depends upon the protein concentration and the divalent ion concentration (Mg2+, Zn2+).

What does alkaline phosphatase buffer do?

Calf intestinal alkaline phosphatase (CIAP) catalyzes the hydrolysis of 5´-phosphate groups from DNA, RNA, and ribo- and deoxyribonucleoside triphosphates. This enzyme is used to prevent recircularization and religation of linearized cloning vehicle DNA by removing phosphate groups from both 5´-termini (1–5).

What is fast digest buffer?

The FastDigest Green Buffer and Thermo Scientific FastDigest Buffer are proprietary digestion buffers which support 100% activity of all FastDigest restriction enzymes. This system allows for double and multiple digestions with any combination of enzymes. No sequential digestions or buffer changes are needed.

When should alkaline phosphatase be used?

Your provider may order an alkaline phosphatase (ALP) blood test to help screen for, monitor or help diagnose liver and biliary diseases, bone disorders and other health conditions. Screening means checking for potential health issues before you experience symptoms.

Which buffer is used for alkaline phosphatase?

For alkaline phosphatase, Tris HCl has been reported as a suitable buffer for maximum activity (Hethey et al. 2002). The effect of buffer concentration on enzyme activity was carried out by assaying the enzyme in Tris HCl buffer in the concentration ranges from 0.1 M to 0.8 M.

What is the optimum pH for alkaline phosphatase?

The pH optimum for alkaline phosphatase activity was 9.0 for all of the isolates tested. The pH optimum for acid phosphatase activity was 5.8 for 67% of the isolates and 4.8 for 33%. All of the isolates showed peak phosphatase activity at 37 degrees C.

What is Tango buffer?

Thermo Scientific Tango buffer, being a part of Five Buffer System, is specially designed for use in double digestions. For more information see www.thermoscientific.com/doubledigest. 1X Buffer Composition. 33 mM Tris-acetate (pH 7.9 at 37°C), 10 mM magnesium acetate, 66 mM potassium acetate, 0.1 mg/ml BSA.

What is the correct time for carrying out the alkaline phosphatase treatment?

4. What is the correct time for carrying out the alkaline phosphatase treatment? Explanation: The alkaline phosphatase treatment is carried out after the cutting of DNA has been done but before the mixing of insert DNA and vector has been done.

Why is dephosphorylation important?

Phosphorylation and dephosphorylation are important posttranslational modifications of native proteins, occurring site specifically on a protein surface. These biological processes play important roles in intracellular signal transduction cascades and switching the enzymatic activity.