Question

 

Lab report for the following topic " Gel electrophoresis for RNA'

The lab report should also have the following criteria Introduction, Aims, Principle, procedure, and results(if possible).

Answer 1

Aim

Separation of fragments of RNA using gel electrophoresis

Principle and reagents used

Gel electrophoresis is the technique of separation of molecules on the basis of their size and charge. It uses a gel which act as a molecule sieve and buffer which provides ions for the conduction of electricity. The separation of molecules is brought about by the application of external electric field.

Agarose gel electrophoresis is used for the separation of nucleic acid fragments whereas sodium dodecyl sulfate polyacrylamide gel electrophoresis is used for the separation of protein fragments.

If we talk about agarose gel electrophoresis, then DNA molecules are all negatively charged because of the presence of negatively charged phosphate group so they will move towards the anode which is the positively charged electrode of gel electrophoresis. Smaller the size of the fragment, faster it will move on the gel and fatherest it will appear from the loading well. Loading well is near the anode of the gel which is negatively charged.

The agarose gel is made by the polymerization of agar which is obtained from red algae. The gel itself act as a molecular sieve for the separation of fragment on the basis of their size. The resolution of the gel is proportional to its concentration. Lesser the concentration of the gel, larger will be the pore size and resolution will be less and vice versa.

The solution of the gel is mixed with ethidium bromide so that when the run of the gel is completed, DNA bands present on the gel can be visualised under UV lamp.

Buffer is used in gel for two purposes. The primary purpose is to provide ions for the conduction of electricity and the secondary purpose is the maintenance of pH.

The samples are loaded on the well by mixing them with loading dye. It is mainly bromophenol blue. It is also called as tracking dye. It is used to track how much of the electrophoretic run is completed.

Procedure

1. Make a solution of agarose gel. Use casting plates to cast the gel. The solution should have ethidium bromide.

2. Pour the solution in between the casting plates. Add some water on the top of the solution which is in between the casting gel.

3. Insert the comb to make wells.

4. Wait for half an hour so that it gets polymerised.

5. Load your samples which are mixed with bromophenol blue.

6. Insert the casting plate along with gel in the electrophoretic unit.

7. Add buffer.

8. Connect the power pack. Switch it on.

9. Let it run of 30 to 40 minutes.

10. Remove the gel and observe under UV lamp.