Question

• Why would one add buffer before removing the comb?
• Summarize how to first obtain a sample via pipette (specifically an adjustable pipette) and then load into the well
• Why is it necessary to use a new tip for each well?
• What factors affect the accuracy of liquid uptake?
• What is the relevance of the difference between the first and second stop?
• Have you pipetted before? Are you guilty of banging the pipette to “firmly” attach the tip?
• What are the applications of gel electrophoresis? In other words, how can it be used?
• How much agarose would you measure to make a 1% solution that consists of 150mL of buffer?
• What is TBE buffer and what role does it have in gel electrophoresis? What is another common buffer that some labs use?
• How did the appearance of the solution change following microwaving? Was microwaving necessary?
• What is ethidium bromide and why is it used? Why is it dangerous?

Answer 1

#1 before removing the combs one should add buffer to the agarose gel so that the change in pH won't affect the sample when it is loaded and run on agarose and the separation of samples can be solely on the sample properties contents of gel do not affect the sample.

#2 in order to load a sample on gel make sure the size of wells that is often written on combs itself and choice of combs depends on type of sample to be loaded and gel pore size. Then according to the well size adjust the pipette and lock it. Now take fresh tip and in air single press the pipette take it inside the sample release the pressed pipette, observe the wells place the tip carefully inside one of the well and release the sample by double complete pressing the pipette.

#3 sample is loaded in micro litres quantity if we will not change the tips they will contaminate each other changing the loading quantity and the bands can not be comparable. So to avoid mixing separate tips must be taken.

#4 relevance of first and stops is to make sure that the sample is not overflow with electric charge as it moves if we are able to see the first stop then all bands are present not a single band is overflowing out of the gel. Secondly, relevance of difference between first and second stop bands are equally moving and have moved in a set to be observed.

#5 gel electrophoresis is the technique to separate the sample (DNA, RNA, PROTEIN) on the basis of size of the fragments in an electric field. It is broadly of two type agarose gel electrophoresis and polyacrylamide gel electrophoresis. When large size DNA fragments are separated agarose is taken and for small DNA fragments and for protein polyacrylamide gel electrophoresis if used , it is vertical.

To compare the fragments size and pattern of different samples gel electrophoresis is used.

#6 for 1 litre of gel 15 grams of agarose is required, so for 100 ml 1.5 g of agarose is required and for 150 litres 2.25g is added.

#7 ethidium bromide or EtBr is a visualising dye it intercalates with nucleotide bases of DNA and visualise the bands in UV. As it intercalates with DNA it is carcinogenic, hence dangerous.