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separation of dna fragments in agarose gel electrophoresis


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7 replies to this topic

#1 deespike

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Posted 01 June 2009 - 09:42 AM

hi all

im trying 2 elute the insert fragment ffrom gel for cloning purpose.the problem is that the insert fragment is around 2 kb and the vector backbone is around 2.1kb.
im trying to run these fragments on 1% agarose gel but the separation is nt much as im nt able 2 distinguish.Is 0.8% agarose gel more suitable than increasing the agarose concentration of the gel.how can i get my insert separated?

thanks in advance

#2 phage434

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Posted 01 June 2009 - 10:45 AM

You may be able to find a third enzyme which cuts the vector but not the insert, making the separation much easier.

#3 HomeBrew

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Posted 01 June 2009 - 01:25 PM

If you can't distingush the fragments in a 1% gel, you'll never distingush them in a 0.8% gel. Maybe go to 1.5% or 2.0%, or use a sodium borate agarose gel, which has much greater resolution that TAE or TBE agarose gels.

If you know the sequence of your vector and of your insert, your best be is to do as phage434 suggests...

#4 aztecan princess

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Posted 01 June 2009 - 03:03 PM

I think it is the opposite, to distinguish between small fragments (ej. 300 and 350 pb), to to a 2 or 3 % agarose gel, but to distinguish large fragments like yours, I use 0.5 % agarose gel and run very slow (it works very well to me). But Iíll go with phage434, look for an enzyme that cut into the vector and not your band of interest, this way youíll have your band and 2 smaller bands.

#5 HomeBrew

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Posted 01 June 2009 - 07:26 PM

Now that I think about it a bit more, aztecan princess might be right -- a lower percentage gel might be better for large molecules. Running the gel slower always results in better separation, regardless of concentration.

#6 deespike

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Posted 01 June 2009 - 08:18 PM

hi

its nt possible 2 cut it any where else coz i cloned my insert into the plasmid with promoter ,terminator and now i want to take my insert out of the vector along with promoter and terminator.i have EcoRV to do so after that i get vector and desired insert fragment.

i think i should try lower concentration of agarose gel

#7 HomeBrew

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Posted 02 June 2009 - 04:28 AM

Do you mean that you need to recover both the vector and the insert fragments intact after EcoRV digestion?

#8 deespike

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Posted 02 June 2009 - 06:43 AM

actually the idea of cutting the vector and making it easier for elution of the insert is brilliant but the thing is that im nt having the enzymes that are in the backbone plus i dnt know whether these restriction sites are present in my gene or nt.to avoid complications i should try for something else.i run my sample in 0.5% gel and in 2% gel also it doesnt separate
can polyacrylamide be used? normal elution kits that elute dna from agrose ,can they do that for polyacrltamide also??




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