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MSc Medical Biotechnology/ Medical Microbiology
“Dry” lab exercises
1. Bioinformatics: 1st exercise
In this exercise, we will use online tools to calculate the DNA fragment lengths that
would result from digesting bacteriophage  DNA with restriction enzymes. You are
already familiar with the first part of this from the BT3004 lab last year, but
everything is else is new.
Navigate to EBI website:
Choose Nucleotide Sequences.
To find the bacteriophage lambda ()sequence: enter the accession number J02459
into the EMBL-fetch box on the left-hand menu. This will bring up the database entry
for the sequence.
Copy the DNA sequence from the bottom of the page (it’s a very long page). It is
about 48000 bases.
Navigate to RestrictionMapper:
Paste the  sequence into the box, and carry out the restriction analysis with enzymes
EcoRI and HindIII.
Calculate what length fragments you would get if you digest  DNA with:
EcoRI plus HindIII
Put these numbers into a table, and save it (you can do the calculations and make the
table using Excel).
N.B. Is  a linear or circular molecule? This makes a difference to how you interpret
the results.
2. DNA Gel electrophoresis (this exercise to be done in your own time, after the
computer class).
The first lane on the left (1, the marker lane) is  DNA cut with HindIII plus EcoRI,
mixed with D cut with just HindIII. The actual lengths of these fragments are
what you calculated from the first exercise.
You will need graph paper for this exercise. You can download log graph paper from
this site:
(i) Plot the distances migrated by each marker fragment, against the log10 of the
fragment length. Note the following:
Agarose gels like this do not resolve (separate) fragments of DNA >20kb.
Fragments that look brighter than they should be, might be 2 coincident or
very close bands.
The smallest visible marker fragment is 564bp.
(ii) Having plotted the calibration curve, estimate the sizes of the DNA fragments in
lanes 2 and 4.
3. Bioinformatics: 2nd exercise
Imagine you have started your lab project. You have been asked to find a homologue
(gene with a common evolutionary origin) for a gene from E. coli in another species
of bacteria, Salmonella enterica, and carry out a PCR amplification of it.
Here is part of the DNA sequence of the E. coli gene:
In order to find the homologues in other species of bacteria, use the BLAST search.
This will compare your query sequence to all other sequences in the database, and
find those that are identical or very similar to it. Navigate to this webpage:
Choose Nucleotide BLAST (blastn) and copy and paste the DNA sequence into the
search box, select the nucleotide collection (nr/nt) database, then click BLAST. When
the results appear, look at the list of matches. Note the species that have homologues
of this gene. Find the one for the species you want (S. enterica, strain CT18) by
scrolling down the page, where your query sequence has been lined up with each of
the matching sequences. Note the nucleotide number at the start of the line labelled
“sbjct” (this tells you where in the S. enterica genome the matching sequence is
located), and obtain the genomic DNA sequence by clicking on the link to the
accession number (blue font, starts with “gi”). Find the CDS (coding sequence, i.e.
gene) that corresponds to the matching region of the genomic sequence. Click the blue
“CDS” link to obtain the DNA sequence of the gene.
(i) What is the function of the protein that the gene codes for? Follow the links and
see what you can find out.
In order to choose PCR primers for analysis of this gene, open a new browser window
and navigate to:
Copy and paste the DNA sequence of the S. enterica gene into the box at the top (the
spaces and numbers will be ignored, so don’t bother to edit them out). There are many
options that can be changed before the sequence is analysed. The only one we want to
change, is to make sure that our PCR product will contain at least bases 100 – 750 of
the gene. Use the “Targets” option on the page to set this and then click “Pick
primers” to run the analysis.
(ii) Record the sequences of the forward and reverse primers the programme has
chosen for you, the size of the PCR product that would result, and note the number of
alternative primer pairs it gives.
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