Question

please answer with explaination

1) You need to purify a 1.2kb fragment away from a 3kb fragment. about what percent agarose gel should you pour? Assuming you need 100 mL to cast the gel, how many grams of agarose should you add?

Answer 1

The separation of DNA on agarose gels are dependent on the size of the fragments to be separated and the percentage of agarose gel prepared. The smaller fragments move faster and tend to club together if the gel percentage is less. For this reason, inorder to separate a 1.2 kb fragment from a 3 kb fragment, we need to use atleast a 1-1.2 percentage agarose gels with 1.2 percent agarose gel being preferred if the DNA mixture has fragments only in this size range.

For preparation of 100 ml of a 1.2percent agarose gel, you need to add 1.2g of agarose to 100ml of buffer ( TAE buffer is used usually), heat it in pulses of 20 or 30 seconds in a microwave oven until the agarose is completely dissolved. The same weight by volume method can be used for any other percentage gel as well.i.e., if you are preparing 100 ml of 1percent gel, use 1g agarose in 100 ml buffer ,use 0.8g in 100ml for 0.8 percent and so on.