1. what is the purpose of using urea in PAGE? 2. During the measurement of the t

Question

1. what is the purpose of using urea in PAGE?

2. During the measurement of the time course, you are asked to take out a small aliquot of the sample. Then you are asked to immediately mix the sample with 10 uL of gel loading buffer. what if you do not do this immediately, say you waited several minutes? what would happen? what is the purpose of adding gel loading buffer?

3. Knowing the single RNA sequence is able to form multiple stable tertiary structures, how does this affect the banding pattern observed on a native acrylamide gel? On a urea denaturing acrylamide gel?

4. How does the rate of the ribozyme cleavage reaction depend upon the reaction temperature? What molecular properties contribute to this phenomenon?

Explanation / Answer

Answers:

1.      Urea PAGE or denaturing urea polyacrylamide gel electrophoresis employs 6-8 M urea, which denatures secondary DNA or RNA structures and is used for their separation in a polyacrylamide gel matrix based on the molecular weight. Fragments between 2 to 500 bases, with length differences as small as a single nucleotide, can be separated using this method1. The migration of the sample is dependent on the chosen acrylamide concentration. A higher percentage of polyacrylamide resolves lower molecular weight fragments. The combination of urea and temperatures of 45-55 °C during the gel run allows for the separation of unstructured DNA or RNA molecules.

In general this method is required to analyze or purify single stranded DNA or RNA fragments, such as synthesized or labeled oligonucleotides or products from enzymatic cleavage reactions.

2.      Loading dye is important for sample run.

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