Monday 19 November 2007

TREATMENT OF NUCLEIC ACIDS WITH ENZYMES

7. TREATMENT OF NUCLEIC ACIDS WITH ENZYMES

(Digestion of DNA with Restriction Endonucleases)

DNA digestion and ligation: chemicals and other reagents were prepared according to the following quantities and concentrations:

5 µl pUC 19 (0.5 ug/ul), 3 µl Hind III, 3 µl of 10 x M buffer, 17µl water and 2 µl bacterial alkaline phosphatase to give a total of 30 µl. Subsequently, a second solution was prepared consisting of 6 µl of 0.5ug/µl of lambda DNA was mixed with 3 µl Hind III, 3 µl of 10 x M buffer and 18 µl of water to give a total of 30 µl also. Both solutions were put in separate micro-centrifuge tubes, spinned down and incubated at 37 degrees C for 2-3 hours. The DNA fragements were checked by Agarose gel electrophoresis that was prepared by 1% gel in TAE buffer.



10 µl of the digestion mixture was picked, 5 µl of sample and 1 µl of loading dye, mixed and applied to the gel. The samples were spinned by centrifugation before application to the gel. Lambda DNA digested with Hind III was used as the molecular marker.


To recover the DNA from the gel, 25 µl of membrane binding solution was added to the reaction mixture and SV mini columns inserted into the collection tube. The mixture was transferred to the mini column assembly and incubated at room temperature for a minute. It was subsequently centrifuged for 1 minute at 15000 rpm, the flow through discarded and mini column re-inserted into the collection tube.

700 µl of membrane wash solution was added and spinned for 15000 rpm for a minute. The flow through was discarded and mini column re-inserted again into the collection tube. This was repeated again once. The mini column was carefully transferred to a clean 1.5 ml tube and 20 µl of water added. It was incubated at room temperature for a minute and spinned down for a minute aft 15000 rpm. The mini columns were then discarded and remaining tubes stored at 4 degrees C or at –20 degrees C.

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