Difference between revisions of "Digesting the DNA"

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(Created page with ' Notes- There should not be too much salt in the digestion because too salt salt will inhibit enzyme activity. Observations: Plasmid Concentration 260/280 ...')
 
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Digestion of DNA is carried out just to look at it (an analytical gel) or, like in this case - cut a band out of the gel for further treatment (a preparative gel). . Typically, a band is easily visible under UV light in an ethidium-bromide-stained gel if it contains about 20ng of DNA.
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Restriction endonucleases such as EcoRI recognize specific palindromic sequences and cleave a phosphodiester bond on each strand at that sequence. After digestion with a restriction endonuclease the resulting DNA fragments can be separated by agarose gel electrophoresis and their size can be estimated.
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Double digestion when you are digesting your DNA with two (or more) enzymes. In such cases, you have to make sure to use the buffer that will be most compatible with all the enzymes. Enzyme buffers are specifically formulated to provide the salt concentration for optimal enzyme activity.
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Notes-
 
Notes-

Revision as of 18:21, 16 June 2009

Digestion of DNA is carried out just to look at it (an analytical gel) or, like in this case - cut a band out of the gel for further treatment (a preparative gel). . Typically, a band is easily visible under UV light in an ethidium-bromide-stained gel if it contains about 20ng of DNA.

Restriction endonucleases such as EcoRI recognize specific palindromic sequences and cleave a phosphodiester bond on each strand at that sequence. After digestion with a restriction endonuclease the resulting DNA fragments can be separated by agarose gel electrophoresis and their size can be estimated.

Double digestion when you are digesting your DNA with two (or more) enzymes. In such cases, you have to make sure to use the buffer that will be most compatible with all the enzymes. Enzyme buffers are specifically formulated to provide the salt concentration for optimal enzyme activity.


Notes- There should not be too much salt in the digestion because too salt salt will inhibit enzyme activity.

Observations:

        Plasmid   Concentration   260/280
        IO500(P)   1790.5µg/µL     1.92
        Lysis(L)   2324.4µg/µL     1.91

For Short Digestion

                DNA = 2µL
 ECORI + Pst1 E1+E2 = 0.5µL + 0.5µL
                BSA = 2µL
             Buffer = 2µL
                 MQ = 13µL
              Total = 20µL

Time taken = 1.5 hours