a)What two restriction sites are you going to use to clone your PCR product into the pL4440 plasmid? What are their DNA sequence? b) State the primer sequence you will use to amplify the F27C1.7 gene ready to be cloned into the pL4440 plasmid? c) How would you go about cloning this amplified DNA into pL4440? Using your knowledge of cloning list 5 important aspects of the method.

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Chapter1: The Human Body: An Orientation
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a)What two restriction sites are you going to use to clone your PCR product into the pL4440 plasmid? What are their DNA sequence? b) State the primer sequence you will use to amplify the F27C1.7 gene ready to be cloned into the pL4440 plasmid? c) How would you go about cloning this amplified DNA into pL4440? Using your knowledge of cloning list 5 important aspects of the method.
From the above sequence I have copied the first 500bp of the
sequence into Word.
ATGGTAAAGGCCGTCGTCGGATTCGGCGCTGCATGCGGAATCT
CTGCTATCGTTGCTTGCCTTTGGGCTGCACTTGTCATCACAAA
TGACATCAATGACATGTATGATGATGTGATGGGAGAGCTCGGA
GGATTCAGAGATATCTCTGATGACACTTGGGGAACCCTTCTCG
ACGTTCGTCACGGAGCCGGAGAGTCTGCTGAGCAATACGTTCG
TGGAATCTTCGGACGTCACAAGCGTTCCAACAGCCAATGCTCT
TGCGGACTTCCATCTCAAGGATGCCCAGCCGGAGCTCCAGGAA
ACCCAGGAGCCCCAGGAGAGCCAGGAGGCACTGGACCAGACGG
AAAGAACGGACCAACTGGACTTCCAGGACTTAACATTCCAATT
CCAAATGACTTCCCTAAGGAGTGCATCAAGTGCCCAGCTGGAC
CACCAGGACAAGATGGACTTCCAGGACAAGAAGGATTCCAAGG
ACTTCCAGGAGACGCTGGAAAGCGTGG
1) You now need to design primers to amplify this 500bp
region of the DNA. Write out the primer sequences in the
correct orientation.
2) Decide on two restriction sites that you can use to clone
this into pL4440's MCS. Identify their sequence.
Tip: The plasmid map is in Figure 3, details of restriction
found at
site
sequences
can
be
https://enzymefinder.neb.com/#!#nebheader
3) Add the restriction site DNA sequences to the correct
end of each primer.
promoter
T7 promoter ccaGCACTGATATCA
TAGATCTAGAA
L4440
Xbal Spel Age! Noo! Nhe!
GGAATTOGATATICA
Apa
Notag
GTÖGCGGCCGCTC
cecAATT T7 promoter
List the steps
required to clone the PCR prduct into the pL4440
plasmid.
Exact recipes and details of PCR mixes + machine
conditions etc are not required, just try and think about
the whole procedure from start to finish.
Transcribed Image Text:From the above sequence I have copied the first 500bp of the sequence into Word. ATGGTAAAGGCCGTCGTCGGATTCGGCGCTGCATGCGGAATCT CTGCTATCGTTGCTTGCCTTTGGGCTGCACTTGTCATCACAAA TGACATCAATGACATGTATGATGATGTGATGGGAGAGCTCGGA GGATTCAGAGATATCTCTGATGACACTTGGGGAACCCTTCTCG ACGTTCGTCACGGAGCCGGAGAGTCTGCTGAGCAATACGTTCG TGGAATCTTCGGACGTCACAAGCGTTCCAACAGCCAATGCTCT TGCGGACTTCCATCTCAAGGATGCCCAGCCGGAGCTCCAGGAA ACCCAGGAGCCCCAGGAGAGCCAGGAGGCACTGGACCAGACGG AAAGAACGGACCAACTGGACTTCCAGGACTTAACATTCCAATT CCAAATGACTTCCCTAAGGAGTGCATCAAGTGCCCAGCTGGAC CACCAGGACAAGATGGACTTCCAGGACAAGAAGGATTCCAAGG ACTTCCAGGAGACGCTGGAAAGCGTGG 1) You now need to design primers to amplify this 500bp region of the DNA. Write out the primer sequences in the correct orientation. 2) Decide on two restriction sites that you can use to clone this into pL4440's MCS. Identify their sequence. Tip: The plasmid map is in Figure 3, details of restriction found at site sequences can be https://enzymefinder.neb.com/#!#nebheader 3) Add the restriction site DNA sequences to the correct end of each primer. promoter T7 promoter ccaGCACTGATATCA TAGATCTAGAA L4440 Xbal Spel Age! Noo! Nhe! GGAATTOGATATICA Apa Notag GTÖGCGGCCGCTC cecAATT T7 promoter List the steps required to clone the PCR prduct into the pL4440 plasmid. Exact recipes and details of PCR mixes + machine conditions etc are not required, just try and think about the whole procedure from start to finish.
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