Molecular Biology-2014
Assignment #5
Exercise 4
Amplification and mutagenesis of LacZ
1. Submit a figure representing your PCR of the LacZ gene. Include an appropriate
legend indicating the expected size as determined by bioinformatics and the
experimental size, determined from your gel.
Exercise 6
Transformation and screening of LacZ recombinants
2. Indicate the number of transformants obtained following the transformation of the
ligation mixtures with and without the LacZ amplicon. Submit your data as colony
counts/mL
3. Submit a figure of the digestions performed on the amplicons obtained by colony
PCR. Include an appropriate figure legend which includes the primers used for the
mutagenesis (PCRmutagen) and the sizes of the undigested amplicons.
4. Submit a table which presents the following information for each of the 4 clones you
screened:
Digestion with XbaI: yes or no
Digestion with XhoI: yes or no
5. According to the results obtained with the above digestions what is the codon
sequence corresponding to the ASP position on the LacZ gene for each of the clones
screened.
6. Indicate whether you would predict that each of the clones screened would cut with
the restriction enzyme BamHI.
Exercise 7
RT-PCR
7. Complete the following table. Indicate whether an amplification product would be
expected (yes or no; theoretical) and whether a amplification product was obtained
(yes or no; experimental):
Source of PCR template
RT reaction of DNAse treated RNA
RT reaction of RNA NOT treated with DNAse
PCR of DNAse treated RNA not treated with RT
PCR of RT reaction on DNAse treated RNA
PCR of RT reaction on RNA NOT treated with DNAse
PCR of RNA NOT treated with either DNAse or RT
PCR without RNA (No template)
Theoretical
Experimental
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Molecular Biology-2014
8. Complete the following table. Indicate whether the source template of the
amplification product is DNA, RNA or both:
Source of PCR template
RT of DNAse treated RNA
RT of RNA NOT treated with DNAse
PCR of DNAse treated RNA not treated with RT
PCR of RT reaction on DNAse treated RNA
PCR of RT reaction on RNA NOT treated with DNAse
PCR of RNA NOT treated with either DNAse or RT
PCR without RNA (No template)
Theoretical
Experimental
Bioinformatics 4
9. Submit the definition, the source organism and the FASTA sequence corresponding
to the accession number NM_000558.3.
10. Submit a table indicating the percentage amino acid identity between each pair of
protein homologs.
11. Submit a table indicating the percentage nucleotide identity between each pair of
nucleotide homologs.
12. With respect to the initial protein sequence, what type of homologue is each of the
other protein homologues?
13. Submit a table which indicates the number of the different types of amino acid
substitutions between the two most closely related proteins.
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