3% NuSieve gel electrophoresis - Why 1kb ladder doesn't move well? (Aug/09/2010 )
I made 3% NuSieve gel
1* TBE buffer 50 ml
,and did electrophoresis twice.
I add 10 microliter of EtBr in the gel tank which is filled with 1* TBE buffer.
I put 20 microliters of each sample(w/4 microliters of gel loading buffer) and 20 microliters of 1kb ladder.
I did this for restriction mapping
But as you can see in the picture which I attached, the 1kb ladder did not move well (the far left lane without the number is for the ladder).
So I cannot tell the size.
Have you guys had the same kind of problems?
Doesn't the 1kb ladder move well in the 3% NuSieve gel?
I don't know what to do.
why did you make the gel 3%? this percentage is useful for separating small dnas. what size range are you looking to separate?
Hi, Thanks for the reply.
1st lane : 1kb Ladder
Lane 1 : 1kb, 3.7kb (SpeI)
Lane 2 : 0.1kb, 1kb, 3.5kb (EcoRI & SalI)
Lane 3 : 0.3kb, 0.6kb, 3.5kb (EcoRI)
Lane 4 : 0.5kb, 3.8kb (BamHI)
I need to do restriction mapping.
So, isn't this good for 3.5kb, 3.7kb, and 3.8kb?
Should I just run the gel for a longer time?
for 3.5, etc, the 1 kb ladder is okay and, yes, you should run it more.
but, for the smaller fragments you may want to use a second ladder which covers that range.