Molecular Biology - Lab Report Example

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The promoter regions are shown in blue. The RNA polymerase III promoter structure refers to the 5S rRNA genes. It is evident that other genes transcribed by RNA polymerase III have different promoter structures, including some with a TATA box and upstream elements similar to an RNA polymerase II promoter…
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Molecular Biology
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Download file to see previous pages Dpn I and Fse I together: fragments of 0.5 kb, 1.1 kb, 1.6 kb and 2.3 kb
Dpn I, Eag I and Fse I together: fragments of 0.3 kb, 0.5 kb, 0.6 kb, 1.0 kb, 1.1 kb and 2.0
kb
a) How many restriction sites are there for each enzyme What, if any, are the unique restriction sites on this plasmid
Ans. Dpn I = 3, Eag I = 2, Fse I = No RS. There are unique restriction sites for Fse I, this restriction enzyme works in conjunction with the Dpn I and Eag I.
b) Construct a restriction map of the plasmid and draw it below.


Cloning Strategies
Question 4 (28%)
Describe outline cloning strategies, including vector types (individual vectors need not be specified) and methods used at each stage, for the following scenarios:
Worked example
You wish to isolate the coding sequence of a human liver enzyme. You have purified the corresponding bovine enzyme and have raised a polyclonal antibody against it.
- Make a cDNA library from human liver tissue - this will be enriched for the
genes for liver enzymes.
- Create the library in an expression vector with a strong promoter so the genes
are expressed in the host.
- Screen the induced expression library for the presence of the desired liver
enzyme using the bovine polyclonal antibody. The antibody will bind to the
colonies which produce the protein they recognise. Although the match may
not be exact there should be enough conserved homology to ensure
recognition.
- Positive colonies will be identified by visualising the label on the bound
antibody/secondary antibody in the colony hybridisation.
a) You have a cDNA clone containing the 900 bp coding sequence of a cell surface
protein from pygmy goat monocytes. How can you use this to find the
homologous cDNA from the merino sheep
b) Having...
The results are as follows:
step. f1 IG SEQUENCE: to make single stranded DNA for sequencing UNIVERSAL PRIMER SEQUENCE: for primer to anneal to, to initiate sequencing SELECTABLE MARKER (eg lacZ'): to allow selection of clones containing the insert MCS POLYLINKER: insert fragment of DNA here 3.0 kb
You must describe the function of the essential features of each plasmid and give some indication of the plasmid size. For expression vectors you must bear in mind the host cells in which the coding sequence will be expressed.
a) Nonsense: The nonsense-mediated mRNA decay pathway degrades mRNAs transcribed from genes in which an amino-acid codon has changed to a nonsense codon; this prevents the translation of such mRNAs into truncated, and potentially harmful, proteins.
c) Splicing: A stage in the processing of mRNA, occurring only in eukaryotic cells, in which intervening sequences (introns) are removed from the primary RNA transcript (hnRNA) and the codig exons are joined together to form the mature mRNA molecule. url:www.geneontology.org .
d) Promoter: A nucleotide sequence of DNA to which RNA polymerase binds and initiates transcription. It usually lies upstream of (5' to) a coding sequence. A promoter sequence aligns the RNA polymerase so that transcription will initiate at a specific site.
e) Reading Frame: A series of triple ...Download file to see next pagesRead More
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