DNA after synthesis were PCR amplified using pFx enzyme...

DNA after synthesis were PCR amplified using pFx enzyme respectively for both premature AI and mature AI. The products of the PCR have blunt ends due to the amplification done by pFx. Mature AI was not able to be amplified using pFx so it was done using Taq Polymerase. PCR clean up of the products were done and was quantified using Nanodrop. Restriction digestion of the Mature AI, Premature AI and pET28-a was done at the Xho and Not1 restriction sites. Purification of the digested products were done using Sodium acetate precipitation protocol. Ligation of the purified products were done using Ligase enzyme. Stage 1 Stage 2 (Cycle 33) Stage 3 95⠁ ° C 95⠁ ° C 55⠁ °C 68⠁ °C 68⠁ °C 4⠁ °C 2:0 min 30Sec 45Sec 1:30min 7:0 min ∞ Table.†¦show more content†¦Pellet was resuspended in buffer and then SDS-PAGE was run for the samples 3.5.5. ProBond purification of Premature AI and Mature AI: Lysis and Suspension buffer composition for BL21 E.Coli cells protein expression are: †¢ Lysis buffer(pH 7.0): 50mM MOPSO, 10% Glycerol, 10mM MgCl2 †¢ Wash buffer(pH 7.0): Add 20mM Imidazole to the lysis buffer. †¢ Elution buffer(pH 7.0): Add 250 mM Imidazole to the lysis buffer. To remove precipitate containing salts column was run (affinity chromatography).In affinity chromatography 1 ml raisin was taken which contained nickel ions. It was equilibrated against tris buffer of pH 8. The equilibrated raisin was added in protein suspension then it was kept for binding for 2 hours. After the binding process, the column was run i.e. affinity chromatography was performed. Histidine tags were present in the protein, it has affinity for nickel ion, and so all Histags got binded with nickel. The elution fractions were collected which contained purified protein and its activity was checked and SDS-Page was run. Before adding resin sample was taken out for assay. After bound sample was taken out in separate tube for assay. About 37 mL of the wash buffer was used to wash the undesired proteins. First two and the last two washes were saved for the activity. About 4 mL of the elution buffer was used for eluting the desired protein. All fractions were