Protocol Online logo
Top : Forum Archives: : Molecular Cloning

Quantifying gel purification products - (May/14/2007 )

Pages: 1 2 Next

Hello,
I have been trying to gel purify a couple of inserts and a pBabe plasmid.
The cloning itself does not seem to be working, and I think it may have to do with insert:plasmid concentration.

The original inserts are in Topo 2.1
Cutting with EcoR1
I am using 1xTAE, ~1% agarose
Montage gel extraction columns

Is there a way to quantify the samples without using most of it?
I have tried using a spec. but I think the TAE messes with it too much for the data to be usefull (even in terms of just builing ratios)

Thanks fr any advice....


david

-olywa510-

I'm new at this.
but I would try precipitating my DNA and resuspending in lower volume of ddH2O. then using a portion for quantification on the spec.
Or, if you suspect it's the plasmid:insert ratio, you could try different ratios like 1:1, 1:3, 1:9. it only requires a few fmols.

-alzaki-

QUOTE (olywa510 @ May 15 2007, 12:29 AM)
Hello,
I have been trying to gel purify a couple of inserts and a pBabe plasmid.
The cloning itself does not seem to be working, and I think it may have to do with insert:plasmid concentration.

The original inserts are in Topo 2.1
Cutting with EcoR1
I am using 1xTAE, ~1% agarose
Montage gel extraction columns

Is there a way to quantify the samples without using most of it?
I have tried using a spec. but I think the TAE messes with it too much for the data to be usefull (even in terms of just builing ratios)

Thanks fr any advice....


david


Did you run another gel to check the quality of your purification product?
Either that or use spectro to quantify. But using gel should be sufficient to roughly estimate how much you have it there.

-timjim-

Run a gel with both the insert and vector, and compare the brightness of the bands. This is typically more revealing than spec measurements, which can often be dramatically wrong (RNA, phenol, proteins, whatever). Of course if you have phenol, you have to get rid of it.

-phage434-

There are also special ladders to quantify low DNA masses (e.g. low DNA mass ladder by Invitrogen). I use this to quantify purified PCR products prior to sequencing.

-kr├╝melmonster-

i compare gel intensities of vector using 1Kbladder which is appropriately resuspended to get half of the mass precised on the datasheet. Then using transilluminator, i compare intensities by measuring the time needed for getting a saturated UV signal.

-fred_33-

I use the 2 log DNA ladder from NEB. Their literature tells you how much DNA is in each band. Its helpful if you want to check concentrations.

-scolix-

we have a standard gel electrophoresis picture and we compare our band with that picture and assume DNA amount

-T. reesei-

Hi,

I feel silly for asking this, but anyway:

Everyone takes pictures of their DNA fragments to quantify?
Aren't you afraid of mutations?

I always cut the band out (on a blue light thingy...) and take the picture afterwards with UV light (there's no camera on the gel-cutting thing).

Thanks,

Moly

-Moly-

QUOTE (Moly @ Jun 6 2007, 07:51 AM)
Hi,

I feel silly for asking this, but anyway:

Everyone takes pictures of their DNA fragments to quantify?
Aren't you afraid of mutations?

I always cut the band out (on a blue light thingy...) and take the picture afterwards with UV light (there's no camera on the gel-cutting thing).

Thanks,

Moly


After purifying the DNA, run 1 or 2 ul of the eluted DNA and have a ladder beside the sample. Now you dont need to worry about the UV light. You have the remaining eluted DNA for ligation.

-scolix-

Pages: 1 2 Next