Hi to everybody,
I am doing the cloning experiment but I have problems before performing the digestion as my clean-up products have a 260/230 ratio of 0.5. There is someone available to explain me how I can improve the ratio getting higher results? Can be a problem of a low concentration after PCR products?
Hi there,
The first data to consider is 260/280 ratio. It should be 1.8 to 2. The 260/230 is 2 to 2.2 for pure nucleic acids. In your case, the low ratio is indicative of the presence of absorbing contaminant. If purifying on silica column the main contaminant is guanidine which is not a problem for digest. If you want to get rid of this contaminant use ethanol precipitation.
Hi there,
The first data to consider is 260/280 ratio. It should be 1.8 to 2. The 260/230 is 2 to 2.2 for pure nucleic acids. In your case, the low ratio is indicative of the presence of absorbing contaminant. If purifying on silica column the main contaminant is guanidine which is not a problem for digest. If you want to get rid of this contaminant use ethanol precipitation.
Davide Caredio How was the concentration of your PCR-product and the ratio of 260/280 ratio before made the cloning.
Giorgia Pertile I did not measure before the clean-up. I only measured after the clean up. Do you thik it is useful to do it before and after? there are some hints suggesting something after PCR-products?
Dominique Liger May you suggest me some protocols to get rid of contaminations with ethanol precipitations? As yesterday I did one but i lost 4 time concentrations with protocol i used. Thanks
Davide Caredio First, did you use a particular kit for the PCR clean up? From my experience, not all kits give good quality for PCR clean up. Also, you will get low DNA concentration for some kits too.
Second, to overcome ethanol contamination, you should make sure your columns or eppendorf tubes are dry with no ethanol remaining after ethanol precipitation. If you are using columns from kits, you can open it (air dry) for about 1-2 minutes after the spinning out the wash buffer which always contain ethanol. If you are not using columns, air dry the tubes after pipetting out the ethanol in the ethanol precipitation step to make sure all remaining ethanol vapourizes out. Most protocols already stress this.
Third, you can also try to do gel PCR purification. Depending on your PCR volume, you can run your sample in a gel (preferably low melting point agarose) and cut out the desired band which will be used for further clean up.
What do you plan to do next if you can successfully clean your PCR product? This will help other researchers advice you appropriately
Davide Caredio I agree with Esther. I made more time the cloning for the qPCR standard and it is important to run the PCR-product (before the cloning) on the gel and after you make the gel extraction. After this make the cloning.
I don't know which is the aims of your cloning, but if you need to make the extraction of the plasmid, I suggest to you the kit of plasmid extraction of Qiagen Macherey-Nagel kit (MACHEREY-NAGEL). This kit present a plasmid more clear without ETOH contamination.
Hi, all.
One other possibility that I already saw happening is water or buffer contaminated. Sometimes, people uses water or buffer which has been left at the benchtop for blanking. After awhile, it may get contaminated with DNA from bacteria or fungi and, as you use it for blanking, spectrophotometers will blank on a DNA solution instead of pure solution (DNA, RNA free ones). I already saw it happening.
Secondly, have you had good results before with the same reaction? Or is it the first time?
Let me ask you: something else
Are you achieving low DNA yields? If so, have you tested and confirmed the amounts by looking them in a gel? You can always check this way and If the amount on the gel did not agree to the spectrophotometer results maybe you're with some contamination on your blanking solution, in the compartment where you put your cuvettes or calibrtation problems in the spectrophotometer. Check it too!
Hope it helps.
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