Ch. 6 Analysis & Characterization of Nucleic Acids & Proteins
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Calculate the melting temps of the following DNA fragments using the sequences only:
a.AGTCTGGGACGGCGCGGCAATCGCATCAGACCCTG CCGCG CCGTTAGCGT
a. (8 x 2°C) + (17 x 4°C) = 84°C
b. (20 x 2°C) + (6 x 4°C) = 64°C
c. (14 x 2°C) + (14 x 4°C) = 84°C
d. (15 x 2°C) + (12 x 4°C) = 78°C
What is the purpose of denaturation of double stranded target DNA after electrophoresis and prior totransfer in Southern blot?
Double-stranded target DNA is denatured to allow hybridization of the probe.
Also,single-stranded DNA binds to nitrocellulose filters.
Name two ways to permanently bind nucleic acid tonitrocellulose following transfer.
To permanently bind the nucleic acid,bake the filter at 80ºC for 30 minutes.
Alternatively, nucleic acid will be cross-linked to nitrocellulose upon exposure to ultra-violet light.
If a probe for Southern blot was dissolved in a hybridization buffer that contains 50% formamide, isthe stringency of hybridization higher or lower than ifthere were no formamide?
The stringency is higher, since formamidefacilitates denaturation of double-stranded DNA.
5. If a high concentration of NaCl were added to a hybridization solution, how would the stringency beaffected?
The stringency would be decreased since high salt concentrations exclude nucleic acids,forcing them close together and promoting hybridization.
6. Does an increase in temperature from 65°C to 75°Cduring hybridization raise or lower stringency?
Increasing temperature raises stringency. Heat promotes separation of the hydrogenbonds holding the two single strands of doublestranded DNA together.
At the end of the Southern blot procedure, what wouldthe autoradiogram show if the stringency was toohigh?
If the stringency is too high, faint or no bands will be present on the autoradiogram.
A northern blot is performed on an RNA transcript with the sequence
The probe sequence is
Will this probe hybridize to thetarget transcript?
This probe will not bind to the targe ttranscript. The sequences are identical and not complementary.
Since RNA does not have a complementary partner, the probe must be complementary to the transcript sequence.
In an array CGH experiment, three test samples were hybridized to three microarray chips.
Each chip was spotted with eight gene probes (Genes A–H).
Below are results of this assay expressed as the ratio of test DNA to reference DNA.
Are any of the eight genes consistently deleted or amplified in the test samples?
If so, which ones?
Gene B is consistently deleted (test/reference < 1.0) and gene E is consistently amplified (test/reference > 1.0).
What are two differences between CGH arrays and expression arrays?
(a) CGH arrays are performed on DNA. Expression arrays are formed on RNA.
(b) CGH arrays detect deletions and amplifications ofchromosomal regions.
Expression arrays detect changes in the RNA levels of specific genes.
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