点击显示 收起
CIMMYT Applied Molecular Genetics Laboratory
1
Quantification and Quality
Control of DNA
UV QUANTIFICATION OF DNA
Add 15 μl of each sample to 735 μl TE, mix well, and read OD260 and OD280 to determine purity. Refer to the
Appendices Section for instructions on how to use the Beckman DU-65 spectrophotometer and for program
listing for automated sample reading.
After UV quantification, adjust the concentration of each DNA sample to 0.3 μg/μl or a concentration of your
choice with TE, and store at 4•C. Sample should be usable for up to 6 months. For longer term, storage at
freezing temperatures is more desirable.
The ratio OD260/OD280 should be determined in order to assess the purity of the sample. If this ratio is 1.8 -
2.0, the absorption is probably due to nucleic acids. A ratio less than 1.8 in-dicates that there may be proteins
and/or other UV absorbers in the sample, in which case it is advisable to reprecipitate the DNA. A ratio higher
than 2.0 indicates the samples may be con-taminated with chloroform or phenol and should be re-precipitated
with ethanol (OPTION B).
A program for the Beckman DU-65 Spectrophotometer is included in the Appendices Section which provides
automated sample entry (with sipper) and calculates all appropriate values for each sample.
DNA QUALITY CONTROL
This step is essential for checking that the isolated DNA is of high molecular weight. For adequate resolution
of RFLPs, native DNA should migrate as a tight band of molecular weight • 40 Kb. However, degradation of
part of the isolated DNA is inevitable, and the protocol below is designed to run the DNA under optimal
conditions for ascertaining the relative amounts of degraded and high molecular weight DNA. The procedure
also allows for verifying the UV quantification performed above.
If you have few DNA samples (say less than 25), check all of them. Otherwise, check only 10-20% of the
samples, making sure that the selection is totally random.
1. Prepare a 10 ng/μl dilution of the selected samples (e.g., 4 μl DNA at 0.3 μg/μl + 116μl TE).
2. Load 100 ng of each diluted sample (10 μl DNA + 2 μl 5X SGB) in a 0.7% agarose gel. Include at least one
lane per comb of uncut Lambda DNA (l) as a molecular weight marker. Load 100 ng (from a 10 ng/μl
dilution) of this marker in order to check both quality and quantity of the sample DNAs.
3. Run the gel at 50 mA for about 90 minutes. See the section on gel electrophoresis for details about gel
preparation, running conditions, and DNA visualization.
CIMMYT Applied Molecular Genetics Laboratory
2
DNA DIGESTIBILITY TEST
This step is essential before setting up large scale digestion experiments. A small amount of DNA is digested
with restriction endonucleases under the conditions described in the next section in order to check the quality
of the digest.
If you have few DNA samples (say less than 25), check all of them. Otherwise, check only 10-20% of the
samples, making sure that the selection is totally random.
1. Put 2 μg of each DNA sample in a 0.5 ml microfuge tube.
2. Prepare a bulk digestion mix based on the recipe given below, and keep it on wet ice. Add 8 μl of this to each
of the tubes containing the DNA. Mix thoroughly but gently and spin down the tube contents.
* Always prepare bulk mixes for the total number of reactions +1 to allow for pipetting errors.
3. Incubate at 37•C for 1.5 to 3h. Stop the reactions with 3 μl of 5X SGB.
4. Load samples in a 0.7% agarose gel and run the gel at 40 mA for 2-3 h. Use Lambda DNA digested with
HindIII as a molecular weight marker. See the section on gel electrophoresis for details about gel preparation,
running conditions, and DNA visualization.