In the experiment im doing we are purifiying Taq DNA Polymerase , and precipitat
ID: 87886 • Letter: I
Question
In the experiment im doing we are purifiying Taq DNA Polymerase , and precipitating it with salts (KCl). After precipitation with PEI , the sample was spin out in the centrifuser for 20 min at 4 degree celsius ,the supernatant was discarded after. then low salt 0.025M KCl was added to the pelletss to resuspend and spun dow again . after the second spin down was the Taq DNA polymerase still in the pellet or in the supernantant and why? what was the pirpose of resuspending the pellet in 0.025 M KCl?
Explanation / Answer
Purifying Taq DNA polymerase:
KCl like any other salt is used for precipitation as you have already mentioned in the query. Then Polyethyleneimine (PEI) precipitation is used for removal of any contaminated nucleic acid. After the centrifuser step the supernatant was discarded because taq DNA polymerase is settled down. After that low concentration of salt 0.025M KCl is added as per your protocol. After the second spin down Taq DNA polymerase is still in the pellet. Because in second spin down we will add buffer solution in the presence of which it will float in supernatant or it can also stick to the walls of the tube and after centrifuser step in second spin down, it will settle down. And it was resuspended in 0.025M KCl which will help in the final steps of purification. The purpose of resuspending the pellet in 0.025M KCl is to act as a buffer solution which can further help in dialyzing the salts(KCl) and protein(Taq DNA polymerase) in the presence of storage buffer. The more the amount of storage buffer as compared to sample which is settled Taq DNA polymerase in our case more will be our dialyzing; which will increase the purification value of the Taq DNA polymerase.