Question

1. Why is it important to keep enzymes at a constant temperature?

2. Why is it important to incubate your samples at 70°C at the beginning of the RT procedure (steps 3, 4)? Why should you immediately transfer them to ice and keep them on ice while setting up the remainder of the reaction? 3. What is cDNA?

"""3. Incubate at 70°C for 5 min *This step denatures secondary structures that might be present in your RNA. Because RNA is single-stranded, bases on the same strand can form bonds folding the RNA and inhibition the reverse transcription reaction 4. Immediately after removing tubes from the 70°C incubation, centrifuge briefly and place tubes on ice. *If samples are allowed to cool slowly, the RNA may refold to form secondary structures.

4. What general procedures in this protocol should you use RNA handling technique, when it this no longer necessary? Why?

Answer 1

Answer 1

Enzymes are active at a particular range of temperature. But maximum activity is shown at a particular temperature known as optimum temperature. Any change from optimum temperature leads to decrease in activity.

Enzymes structure is highly sensitive to change in temperature. Large increase in temperature can cause enzymes structure to disrupt, thus leading to inactivity of enzyme. Due to large increase in temperature enzymes starts unfolding their structure because of breakage of hydrogen bonds that make up the three dimensional structure of enzymes.

At low temperatures enzyme activity is very low because substrate molecules have very less kinetic energy.

Therefore enzyme temperature should be kept constant.