PRACTICAL 4
Materials and Methods
Measurement of DNA concentration
The most common technique to measure DNA concentration is measurement of absorbance. We had used 1:20 dilution of the DNA sample and the reading was expected to be in the range of 0.1-1.5 OD260. 5µl of DNA and 95µl of PBS buffer were mixed together and inserted into a clean cuvette. Then it was put inside the spectrophotometer. The measurement was taken at 230nm, 260nm and 280nm. The DNA concentration was calculated by using this formula:
DNA (µg/ml) =OD260 x 50 x dilution factor
For more accurate reading, we had used NanoDrop™ which directly gave us the DNA concentration.
Digestion of DNA with Restriction Endonucleases
Two eppendorf tubes were prepared for digestion of plasmid and SOD gene. For plasmid digestion tube, 6µl plasmid DNA, 2µl buffer R, 1µl BamHI, 1µl HindIII and 10µl distilled water were added. While, 5µl of purified PCR product, 2µl buffer R, 1µl BamHI, 1µl HindIII and 11µl distilled water were added into SOD gene tube. Next, 1 µl of 0.2mg/ml RNase was put inside both tubes and spinned by using microcentrifuge for a few seconds. Those tubes were then placed into 37ºC water bath and incubated for 2 hours. The tubes were deactivated by heat at 65ºC for 10 minutes and stored in -20ºC.
Results
Measurement of DNA concentration Equipment
Optical
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The result has shown by using Agarose Gel Electrophoresis (AGE). There are two possibilities outcome for digestion of pUC19 and SOD gene which is positive and negative result. Positive result should only have a single band formation at specific length while negative result will show few bands on varies length and contamination by other components. All groups have shown one band formation for digestion of SOD gene at 550bp in length while only group 11 has successfully obtained a single band formation at 2686bp for digestion of pUC19 by restriction