I need to know how much DNA I have in my samples after doing the PCR, does anyone know the mode for measuring using a nanodrop? What can I use as a target? The mix? The other reagents??
any suggestions?
Thank you.
Rodolfo
The high concentration of nucleotides will not allow spectrophotometric analysis of PCR products. Use a kit to purify PCR products, then nanodrop.
The pcr products are masses of various amplicons. if you want to check your target, you need to do electrophoresis with gel. but I think its yield is not guarantee to use for quantification. I recommend to use Digital PCR.
NanoDrop is based on absorbance and the OD that is obtained is used to calculate the concentration of DNA present in it. One can measure the concentration of DNA using PCR product also. the only thing that should kept in mind is that a blank should be taken that would the the PCR mixture that has been not kept for amplification.
Good morning, Dear Rodolfo.
To quantify you DNA after PCR, you should purify your PCR products. As you realized, if you want to quantifty immediately after the PCR is done, there are a lot of interferences (all the left components of the mix) that won't help us to obtain a feasible result.
Until now, I've only used two ways to purify Pcr products. First, running in a gel and then cutting the section of our amplicon. And the second is employing a filter system (similar to the kit of DNA extraction) when you're PCR reaction is done. However, to assure your results, I suggest you to purify first.
Also, this choose will depend on the future purposes of your DNA (sequencing, cloning, viral load,...). Hope it helps, if you still need suggestions, you can leave me a message.
If you have a Real Time PCR, you can do Absolute Quantification to solve this trouble.
If you use Nanodrop with flourescent dye (Nanodrop3000) it could work.
Otherwise it is necessary to purify. You cannot creat a good "blank".
For purification you can either go for Agarose gel and cutting out (like others recommend) or use other kits. I used two, one worked okay the other not.
Another option is to measure the concentration of you PCR product by taking a fotograph of your agarose gel and than applying a image analysing software. The ladder mix serves as standard.
Many Thanks to all, the suggestions generally indicate that purification is the next step if I want to quantify my DNA. The objective is to have a sufficient amount of DNA for sequencing. Will cut the part of the gel with my fragment and will use purification kit.
:D
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