Gel purified samples not sinking in gel well's - Not getting my samples to fall to bottom of well (Aug/03/2006 )
I purified a band from my gel with the Wizard Gel purification kit and then loaded it on a gel, but it didn't sink to the bottom of the well. I think i might be getting some ethanol residue. I am centrifuging it an extra 2 minutes after the last wash and am eluting with 25 uL of preheated water. Any suggestions?
Definitely sounds like EtOH, but you may also want to try adding additional glycerol or sucrose to your loading buffer or sample before running it.
I have had this probem too. If you add an excess of 1 M tris, the DNA will stay in the wells. I cannot tell you exactly how much. I just added an equel vol. 10ul DNA + 10ul tris, but this probably overkill...
Yes, I always experience this problem with the gel extracted samples.
So, most of the time I will just add more loading dye to the sample.
Further, for the gel extracted samples, most of the time when I spec it with nanodrop spectrophotometer, the concentration of the nucleic acid given by the machine is not very accurate. There was once, it showed 0 ng/microL. However, when I resolved a small amount on gel, I could see my product. Any explanation???
Does anyone think eluting with TAE buffer instead will make a difference. I know that one or two times I haven't had this problem but don't recall doing anything significantly different.
That sounds strange.
I use quiaquick spin columns from qiagen, and I never had problems.
In the handbook, they say that you have to discard the flow-through,after the wash step, and centrifuge the column for an additional 1 min to remove completely the residual ethanol.
this will not be achieved if you do not discard the flow-through before this additional centrifugation.
Did you do this step?
I actually spin it for two minutes after discarding the flow through then discard the flow through and spin another minute and it did it again today. My dna usually shows up alright but I don't know if I am getting a good idea of concentration.
Sorry, I just realize you wrote at the beginning of the post that you did the additional centrifugation.
I don't know what to tell you beside adding more glycerol.
i use the PCR purification Kit from Amersham and i never had that problem!
one student had once that Problem when he used the 50x TAE instead of the 1x.
if u use 6x Loading Dye try to add more or add more Glycerin to it...or use the same volume of 10x.