DNA loading buffer - (Oct/27/2006 )
I did a DNA loading buffer for agarose gele to load my smaple.
I got this recipe online. the thing is when I load it looks like my sample does'nt go in the bottom of the well but go away from the well and I lose it.
Iwas wondering if it's the SDS. is it normal that it has SDS in it?
thanks
Dissolve in 6.25 ml of H2O
* .025g of Xilene cyanol
* .025g of Bromophenol Blue
* 1.25ml of 10% SDS
* 12.5ml of glycerol
This phenomenon is very common when you have EtOH in the DNA. Are you sure you don't have EtOH?
I'm not sure if it is the SDS causing problems, but your recipe is similar to the one we use:
9mg Bromo blue (0.09%)
9mg Xylene Cyanol FF (0.09%)
8.8mL 60% glycerol
1.2mL 0.5M EDTA
In the past, we had about a week where all of our wells failed to load due to inadvertantly having residual ethanol in the pellet resuspension (after mini-preps). Not sure if this is your situation, but you may want to look into that.
Otherwise, I would try making up a small batch of 6X loading dye with a different recipe and see if this corrects the problem.
@ dnafactory
lol...if I could type faster I wouldn't have had to include this in my comment!!
I never thought about the EtOH, that might be the reason now that I think about it. I had the same problem once, and I solved it with 6X loading buffer.
I wouldn't put SDS in a DNA loading buffer. It's negatively charged so will move through your gel
I use (or used)
40% sucrose
5X TBE
a dab of orange G or bromophenol blue
You make this by adding 5ml of 10X TBE, 4g of sucrose and make up to 10ml with nuclease free water. Then dip a pipette tip in the dye powder and add to the clear buffer until it gets to the desired colour
Use as 5X - this way the sample in the well is the same TBE conc as the gel
I use (or used)
40% sucrose
5X TBE
a dab of orange G or bromophenol blue
I like the dipping a pipet tip into the powder to get just a little amount. Can you tell me about what band size the orange G runs at?
On a 1% agarose gel I think it's about 20-50bp but I'd have to check.
Thanks! With my recipe I think the Xylene runs about 500bp and it tends to obscure some of my bands every once in a while. I may have to use some of the orange G to clear this up!
thanks all
jm