Size standards and loading dyes

Size standards

We include a size standard on every gel. This is typically 1 kb plus from Invitrogen (10787-026). This has 12 bands from 100 bp to 12 kb. To quantify DNA concentration, you can also use a DNA mass ladder where intensity is proportional to DNA concentration.

It is optimal to load 0.5 ug of size standard in a gel. If you make up the size standard in loading dye to the following recipe, load 10 ul to get 0.5 ug.

Size standard recipe

	1 ug/ul size standard	0.5 ug/ul size standard
Size standard (10 ug)	10 ul	20 ul
Loading dye	40 ul	40 ul
Water	150 ul	140 ul

Loading dyes

We use several dyes. Orange G runs right at the front (at <50 bp) so you never run your sample off the end of the gel. It can be loaded from parafilm. Blue dye can be made up either with just one dye (xylene cyanol) or with two (xylene cyanol + bromophenol blue). These dyes run at different equivalent DNA sizes depending on the gel %. They can quench the fluorescence from DNA which co-runs with them. I typically dilute the dyes so they aren't so strong.

Orange G recipe (5x)

	Amount
Glycerol	5 ml
0.5 M EDTA	1 ml
2% orange G	1 ml
10% SDS	0.1 ml
water	2.9 ml

2% orange G can be made from 0.2g in 10 ml of water. Orange G is available from Sigma.

Xylene cyanol (6x)

	Amount
Xylene cyanol (0.25%)	25 mg
Sucrose (40%)	4 g
Water	10 ml

To make diluted xylene cyanol add 1 ml of 6x dye to 10 ml of 40% sucrose (4 g sucrose in 10 ml water).

 
KC 9/01