DNA Gels, Blots and Probes Flashcards
How can DNA fragments of different lengths be separated?
Gel electrophoresis.
Where is the digested DNA sample placed?
Into a well in a porous polysaccharide gel immersed in a buffer solution.
Where does the electrical current run?
Runs between the two electrodes placed in the buffer.
Where does the DNA move and why?
To the positive electrode because it is negatively charged.
What causes the fragments to separate along their lane according to their size?
The charge is uniformly distributed along the sugar phosphate backbone of the sugar-phosphate backbone of the molecule. The smaller fragments move faster and further through the gel.
What might a well in a gel usually contain?
A DNA ladder.
What can a DNA ladder be used for?
It displays a selection of known lengths of DNA that can be used as a reference scale.
How can DNA become denatured?
Heating it up to approximately 95 degrees C, braking the H bonds holding the complementary bases together
What is annealing or hybridising DNA?
The formation of a double-stranded molecule from single strands.
Why is the melting point of DNA with a high proportion of C:G pairs higher?
Because C:G pairs have more hydrogen bonds
Why are DNA blotting procedures used?
To make an accurate record of the final positions of the DNA fragments after electrophoresis.
What are blotting procedures used to do?
Used to draw the DNA fragments out of the gel and onto a nitrocellulose or nylon filter.
Why is the DNA denatured chemically with sodium hydroxide before the procedure?
To enhance the attachment of DNA to the filter.
What are DNA probes?
They are short single stranded sequences of DNA that have been labelled either radioactively or with a fluorescent tag.
What is specific about the design of a DNA probe?
Their sequence is designed to be complementary to a DNA sequence.
