3 replies to this topic

[Eternal city]

Hi guys,

I made 3% NuSieve gel
   Nusieve 1.5g
   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.


Thanks,

JA

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Edited by Eternal city, 09 August 2010 - 11:42 AM.

[mdfenko]

why did you make the gel 3%? this percentage is useful for separating small dnas. what size range are you looking to separate?

[Eternal city]

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?

Edited by Eternal city, 10 August 2010 - 06:23 AM.

[mdfenko]

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.