Accurate quantification of the template prior to sequencing is critical. Depending on the amount of DNA used, several different methods are available. Two other methods are described below: fluorescence assay and comparative bromide staining. This experiment is from PCR Lab Guide (2nd edition) by Kang Seed and Lijia Qu.
Operation method
PCR product quantification
Materials and Instruments
DNA Standard TE PCR products Move Method A: Fluorometric method For more product details, please visit Aladdin Scientific website.
Fluorometers, foil, microtubes
Fluorometric methods allow for accurate quantification in the nanogram range. Fluorometers and DNA quantification kits are available from many companies. It is important to determine if the fluorometer is fitted with components appropriate for the excitation and emission of a particular dye. The method is designed to work with micro-incinerators equipped with filter devices with blue LED light sources, microcell attachments, tubes (TurnerDesigns), and PicoGreen dsDNA Quantitation kits (Molecular Probes).
I. Materials
1. Buffers and solutions
LambdaDNA Standard (100ug/ml)
TE (10 mmol/LTris-HCl, lmmol/LEDTA, pH 7.5)
2. Nucleic acids and oligonucleotides
Purified PCR products
3. specialized equipment
Fluorometer, foil, microcentrifuge tubes
4. Other
PicoGreen dsDNA Quantitation Kit (MolecularProbes, P-7589)
II. Methods
1. Prepare the standard concentration of DNA: Dilute lul of standard λ-DNA (100ug/ml) into 49ul TE, so that the final concentration of DNA is 2ng/ul; take another 3 tubes, add 60ng, 20ng and 2ng of DNA respectively, and make up to 50ul with TE; and then prepare a blank control by adding 50ul TE.
2. Prepare the sample: usually 0.5ul or less of PCR purified product is enough, make up to 50ul with TE.
3. Dilute PicoGreen reagent with TE1:200, then wrap it with aluminum foil to avoid light, add 50ul of diluted PicoGreen to each DNA sample and blank control, mix well, and leave it in dark condition for 5 min.
4. Read the blank control with a fluorometer and calibrate with the highest concentration of standard DNA, then measure the other standard samples, test the linearity of the calibration, and then measure the samples.
Method B: Comparison of Bromide Staining Methods
If a fluorometer is not available, the concentration of the PCR product can be estimated by agarose condensation electrophoresis, and the PCR product can be quantified by comparing the brightness of the bromide-stained bands with a known amount of DNA. High- and low-DNA-volume gradients (Invitrogen, 10496016 and 10068013, respectively) are useful DNA volume standards that contain equal amounts of each DNA fragment, with high-molecular-weight standards ranging from 1 to 10 kilobases (kb) and low-molecular-weight standards ranging from 0.1 to 2.0 kb.