Q1
Draw, on the template gel provided below, the banding pattern you would expect to see when plasmid pET21a is digested with the following enzymes:
Lane 1 = EcoRI alone
Lane 2 EcoRI plus PstI
Lane 3 EcoRI plus PstI plus BamHI
Q2 10 marks
Go to http://nc2.neb.com/NEBcutter2/ . The file is in fasta format, as indicated by > on the top line.
Paste the sequence:
AY948115.1 GAGGGCGACAAAAGGGAACAGACCCAAAACCACAGGAGAGATGCTAGCATGACAGGGATGCAGAGACATA
AAGCACAACAGTGAGATGGAGTTAATATACCTCCACGAGGGTGACCTTGTCCTGCATCTCAAATTTTGGG
TAGGATTTGAATGGGCCAGAGGGACAGAAAAGAAGAGAAAGAGCATGATGAGCAAGGGCTTGAATGTTAA
ATAGATTCCTCTTTGGGGGACCAGGGAGATACAAGCTTCTAAAGCACATACGCCCTGTATTGGAGAATGG
GGAGGAGTAGATAGATGAGAAGGTTGAAGCCATATTACGAAGCCTTGAATGCTGAACATCAGATCTGGGG
CTATATTCTTACCTTGATACATTTCAGAAGCAACTGAAATCGTAGGACCTTCCTTGCTTCTCTATTGGGT
GAATGTTTCTCAGTCTTGGTGTGAGTCTCAGTGCCTACGTAGTTAAAGCTTACTGAAATGTTCCCTTTAC
AATTCTAGAGAGATATGTCCTTTATGTTGACATGTTCATGCTGACAGACTGCATCTGATTAAACAGCTGC
CTGTGCAATGCCTCCAAGTGTGGATAAAAGAAAAATTAAACTCATAATCTTGGACAGCCATGTGTAGACT
AGTTACATTGATCAAAGGGCAATAGAAATGATCCAGTGAGGATTTGTCTGAATTTCCCACAATTATTTAA
AATCTACCTCAAATACCTGTTCATCTATAATGCCTCCCCTGAGGCCTTCATTCTGAATAGTACCTCTGTC
TCTGTCCCCAAAGCACTAACTGATCCCTGTGATAGCGCACTTCCCAGCCAGGCTGATATGTAGACTTGGC
TGCCTGTGTATCTTTTCCCCATAGACTGTGAGCTTCCTTTTATGAATAATAATTGTAGCTAGCATTTAGT
AGGGTGCTCCTACCTGTTAAACTCTATGATGAGTGCTTTACATAGATTATATCATTTATTCACTAAACAG
TCCTTTAAAATGGTGCTATATTCACTAAACAGTCCTTTAAAATGGTGCTATATTCACTAAACAGTCATTT
AAAATGGTATTATTCTTCTTCATCTTACAGGTAAACAAACTAAGGCAAAAAAAAAAGTGAAATAATAAGT
GCCAGTACACAGAGCTAGTAAGGAATAGGGTCTGCCAGGTCCCAAAAAGCATGCCATCACCTTTGCCCCA
TACTGCCTCTGGTACAGATAGAGGTAATGTCTTATTTATCACTGCCATCCACTGGACCCAGCTTAGTGCC
TGACACACAGAGGGGCTCAGTCAATGCTGATTGGTTTGAGGTGGAGCAAAAATGCTTAGCAGGGTGAGCA
CCTTTGCTGTGATTGAGTATCTGATTCTCTATGAAGAGAAGGGGAGTCCTGAGCCAAACACATTCCTCTG
GCTCCTGGCTGTCATCTTTATTTGCCCGGCTTCTTTGCTCTTCCTCCTTCCTAACTGCACCGTTTGGATT
CAAAGCTGGAGCTTAATGCAGATAAAGGGAAAACAGAACTTTGAATGACCACTGTGGGACTAAGAGAGGA
GAAACAAGAAATTTGACAGATGAGGAATAAAGTGAGGAGAAGAGAAAATGATTAAGCTTTATCACTTTAA
CTTAATATTTAACCTAATGAAAACAAAATCTTATTTGAAATTGGAAAAATCAATGTATTGATTGCTGGTT
CATTGCCCTCTTCTTTATGATTTGACAGTCTGTGAATAATCTAATGGGTGTGGCTTAAAGACCTAGATCA
TGTGTGGAACTGGAATCGGGTGTTATTCAAGCAAAAAAAATAAATAAATACCTATGCAATACACCTGCTT
TATGCACTTGAGCAGGGAAGAAATCCACAAGGACTCACCAGTCTCCTGGTCTGCAGAGAAGACAGAATCA
ACATGAGCACAGCAGGAAAAGTAAGCAAAAAATATATTACTGTTGGAACTATATTCTCATCAATATAACA
AAGAAAGTAATACAGTATTTGATGAATCATTTAAAATTCATATCTAAATTAGAAATGATACACTGAAATA
TGATATGCAATATATGCTATAATATGTAATGTATACTGAACTACAGTGGAAATAAGCTATTCCTAAATAC
CTTCAAAAAGAATGTATAGAATCTGTATCTATGAAGTGTTTATTTCCCACATTAAAGACATTTGCGGTAA
AGCGATAATTTATTCCAAGCTAATCATGATTAATTTGTAAAGCCAAAGTTAGAAATGTCTTTCATCAAGA
AGTTTTCTTTATATTAAGGTACCATAATTTAAATGTATTACTATTTGTATTTATATTTTTTGTGAATACA
(including the first line beginning >) into the search box.
Ensure that ‘The sequence is linear’ is selected.
Submit the sequence. A complete and complicated restriction map of the DNA sequence will be returned to you. Simplify the map using the Custom digest option.
In the template below draw the banding pattern you would expect when the DNA in question is restricted with
(a) Xba alone
(b) PstI plus BglII
Q3 10 marks
Imagine that gene x (3000 bp) is cloned into pUC18 as a HindIII-KpnI insert to create the recombinant plasmid pUC18 X. pUC18X is then transformed into E.coli DH5α cells.
Briefly describe the rationale of plate screens that would differentiate between untransformed cells, cells containing re-ligated pUC18 and cells containing pUC18X. You are not asked to describe the underlying principle of the plate screen or to give technical details.
Q4 10 marks
Describe the restriction digests you would use to demonstrate that gene x (see Q3) had been successfully inserted into pUC18 between the HindIII and KpnI sites. Include at least one control digest in your scheme. Draw the expected banding pattern in the gel template below.
Q 5 10 marks
Draw a Plasmid Map of pETrb3. Follow the instructions given in your practical manual. Indicate the rb3 insert on your map. It should be clear where the insert starts and ends.