Question

Discuss in detail about demonstration of alkaline phosphatase

Answer 1

Answer:

Thermo Scientific FastAP Thermosensitive Alkaline Phosphatase catalyzes the release of 5'- and 3'-phosphate groups from DNA, RNA, and nucleotides. This enzyme also removes phosphate groups from proteins.

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. The enzyme is inactivated in 5 minutes at 75°C (see Figure 1 in Supporting Data). Therefore, removal of alkaline phosphatase is not required prior to ligation.

Highlights

• Recombinant enzyme

• Fast dephosphorylation—10 minutes at 37°C

• Fast and complete inactivation—5 minutes at 75°C

• Simultaneous digestion and dephosphorylation of vector DNA

• 100% active in restriction enzyme and PCR buffers

• PCR clean-up in conjunction with Exo I

• Protein dephosphorylation

One protocol for all types of DNA ends:

• 5'-overhangs

• 3'-overhangs

• blunt-ends

• single nucleotides