Estimation of quantity and quality of isolated DNA

Methods for Determining DNA Yield and Purity DNA quantity and quality can be assessed using several different methods include: 1-Absorbance by spectrophotometer or Nanophotometer. 2-Agarose gel electrophoresis . The most common technique to determine DNA yield and purity is also the easiest method—Absorbance

NOTES: Nucleic acids absorb light at 260 nm ,the A260 reading should be between 0.1–1.0. Note that the spectrophotometer is most accurate when measurements are between 0.1–1.0. However, DNA is not the only molecule that can absorb UV light at 260nm. Since RNA also has a great absorbance at 260nm will contribute to the total measurement at 260nm.

Quantification of DNA by spectrophotometry Using TE buffer as the diluent, make an appropriate dilution of your DNA depending on the size of the cuvettes available (e.g. for 1ml cuvettes, dilute 10 microliter DNA solution in 990 micro liters of TE). Determine the absorbance of DNA at 260 nm using TE as the reference solution (i.e. as a blank)

Using a conversion factor : one optical density unit (or absorbance unit) at 260 nm is equivalent to 50 microgram/mL of DNA and 40microgram/mL of RNA. Multiply the absorbance reading by the conversion factor and the dilution factor to find the concentration of nucleic acid.

Pure DNA Concentration (microg/ml) = (A260 reading – A320 reading) x dilution factor x 50microg/ml Total yield is obtained by multiplying the DNA concentration by the final total purified sample volume. DNA Yield (microgram) = DNA Concentration x Total Sample Volume (ml)

Note A reading at 320nm will indicate if there is turbidity in the solution, The 320 nm absorbance is used to correct background absorbance. (BLANK)

Example 1. A DNA preparation diluted 1:100 yields an absorbance reading of 0.200 at 260 nm. To obtain the concentration in micro gram/mL: 0.200 absorbance units x 50 microg/mL x 100 =1000 microg/mL The yield of the sample is calculated using the volume of the preparation. If in the case illustrated above, the DNA was eluted or resuspended in a volume of 0.5 mL The yield would be: 1000 microg/mL x 0.5 mL =500 microgram

Quality of DNA using spectrophotometer DNA UV absorbance at 260 nm. protein UV absorbance at 280 nm . The ratio of the absorbance at 260 nm/280 nm is a measure of the purity of a DNA sample from protein contamination; it should be between 1.7 and 2.0 The absorbance of the nucleic acid at 260 nm should be 1.7–2.00 times more than the absorbance at 280 nm.

If the 260 nm/280 nm ratio is less than 1 If the 260 nm/280 nm ratio is less than 1.7, the nucleic acid preparation may be contaminated with unacceptable amounts of protein and not of sufficient purity for use. Such a sample can be improved by reprecipitating the nucleic acid step of the isolation procedure DNA Purity (A260/A280) = (A260 reading – A320 reading) \ (A280 reading – A320 reading)

A DNA preparation with a ratio higher than 2.0 may be contaminated with RNA. If RNA may interfere or react with DNA detection components, RNase should be used to remove the contaminating RNA.

The ratio of the absorbance at 260 nm/230 nm is a measure of the purity of a DNA sample from organics and/or salts; it should be about 2.0. Low A260/A230 ratio indicates contamination by organics and/or salts

Some notes about Nanophotometer Don’t need dilution The volume required for measurement 3-5 microliters The concentration given in nanogram \microliters.

Quality from Agarose Gel Electrophoresis Quality of DNA extracted is assessed using the following simple protocol: Mix 5 µL of DNA with 5 µL of loading Dye Load this mixture into a 1% agarose gel Stain with ethidium bromide Electrophorese at 70–80 volts, 45–90 minutes.

Range of separation of linear DNA Agarose (%) Range of separation of linear DNA (in kilobases) 0.3 60 - 5 0.6 20 - 1 0.7 10 - 0.8 0.9 7 - 0.5 1.2 6 - 0.4 1.5 4 - 0.2 2.0 3 - 0.1

DNA Quality from Agarose Gel Electrophoresis High molecular weight band (>48.5 kb) Smearing indicates DNA degradation (or too much DNA loaded).

DNA Quality from Agarose Gel Electrophoresis High molecular weight band Smearing indicates DNA degradation

DNA Quality from Agarose Gel Electrophoresis Lambda DNA marker Human Whole Blood DNA Lambda DNA cut with Hind III marker Whole blood genomic DNA

Troubleshooting Nucleic Acid Preparation Methods Problem: No or low nucleic acid yield. Make sure that ample time was allowed for resuspension or rehydration of sample. Repeat isolation from any remaining original sample . Concentrate dilute nucleic acid using ethanol precipitation.

Poor DNA yield 1-The blood sample may contain too few white blood cells. Draw new blood samples. 2- The white blood cell pellet was not resuspended thoroughly . 3- The blood sample was too old. Best yields are obtained with fresh blood. Samples that have been stored at 2–8°C for more than 5 days may give reduced yields. 4- The DNA pellet was lost during isopropanol precipitation. Use extreme care when removing the isopropanol to avoid losing the pellet.

Troubleshooting Nucleic Acid Preparation Methods Problem: Poor nucleic acid quality If sample is degraded, repeat isolation from remaining original sample, if possible. If sample is contaminated with proteins or other substances, clean it up by re-isolating repeating the protein removal step of the isolation procedure.

Degraded DNA (<50kb in size) Improper collection or storage of the blood sample. Obtain anew sample under the proper conditions.