Question

Double digestion of the the CAN1.Z ORNA and the PMD2 plasmid Bola I-HF SCHI CANL.Z! CANL.Z -SHI ©- MD2 CANLY → PMD2 CANLLY Bok I-HF You will cut both the plasmid PMD2 and the CAN1.Z ORNA using 2 enzymes (Bstell-HF and Sphl). These are two double digestions that will leave ovehangs that are compatible between the digested plasmid and the digested ORNA. You need to set up 2 digestion reactions (total reaction volume: 504L): A. Plasmid PMD2 + Bstell-HF + Sphl B. GRNA amplicon (from your PCR of OMD19) + Bstell-HF + Sphl → For this digestion experiment, you will not set up reactions for the negative controls. You will have one negative control for the PMD2 plasmid (=undigested plasmid) and one negative control for the CAN1.Z ORNA (=undigested ORNA). Instead of setting up reactions for these two negative controls you will just prepare (next week) the undigested samples and run them on the electrophoresis gel. Materials Enzyme buffer (Cutsmart) Restriction enzyme: Bstell-HF (incubation 37°C: 4U/L) Restriction enzyme: Sphl (incubation 37°C: 4U/L) Plasmid DNA (50ng/L) miliQ H2O Ice bucket
What would happen if BsteII-HF had an optimal temperature of 30°C but we used the same double digestion protocol as we used in lab 4? (2pts)

Note: consider all the cloning steps for this question

Answer 1

Restriction enzyme in sub optimal temperature results in star activity. If we digest the DNA with both enzymes ( double digestion) at same temperature (37 degree celcius), the digestion may result in star activity ( non specific recognition of restriction sites that result in improper digestion ) since BsteII-HF enzyme has optimum temperature of 37 degree celcius