Gel Electrophoresis - loading dye and more

Question

I'm a student new to molecular biology coming in from an eco-evo background looking for tips from you molecular pros! It seems that I'm running quite a few gels. The loading dye I'm using is Gel Loading Dye, blue (6X). The supporting document calls for 5uL:25uL reaction and 10uL dye:50uL reaction. Here's the problem: I'm trying to get my PCR to work and so I'm doing a lot of 'just checking' trials where I only load 2uL of reaction to each well from different 20uL PCR reactions. What volume of Gel Loading Dye, blue (6X) should I use when running only 2uL of PCR product

My goal now is to learn to do a DIG in situ hybridization to localize the distribution of mRNA of a gene I'm interested in so any tricks and tips would be greatly appreciated!

Answer 1

The loading buffer you add to your samples for gel electrophoresis has a few different purposes, but the exact amount does not really matter. The purpose of the loading buffer is

• to make the sample heavier so it is easy to get into the pocket and stays there
• to visualize how far the gel has run
• to denature the sample (only for denaturing gels)

Sample buffers for agarose gels usually use something heavier than water like glycerol or sucrose together with a tiny amount of a dye like bromophenol blue.

So in general there is no issue with using too much dye, but using not enough could lead to the sample being not heavy enough and problematic to pipet, or the sample not being properly denatured. I'd either use 1 ul of the sample buffer (smaller amounts can be annoying to pipett) for your sample, or dilute the sample to 5-10 ul and use the regular amount.

Answer 2

When using small sample volumes, it is advisable to use water to increase the volume and make handling and mixing with loading dye easier. For example, you may mix 2 uL of your sample with 1 uL 6X loading dye and 3 uL water for a final volume of 6 uL.