1. What two restriction sites are you going to use to clone your PCR product into the pL4440 plasmid? What are their DNA sequence? 2. State the primer sequence you will use to amplify the F27C1.7 gene ready to be cloned into the pL4440 plasmid? 3. How would you go about cloning this amplified DNA into pL4440? Using your knowledge of cloning list 5 important aspects of the method.
Molecular Techniques
Molecular techniques are methods employed in molecular biology, genetics, biochemistry, and biophysics to manipulate and analyze nucleic acids (deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)), protein, and lipids. Techniques in molecular biology are employed to investigate the molecular basis for biological activity. These techniques are used to analyze cellular properties, structures, and chemical reactions, with a focus on how certain molecules regulate cellular reactions and growth.
DNA Fingerprinting and Gel Electrophoresis
The genetic makeup of living organisms is shown by a technique known as DNA fingerprinting. The difference is the satellite region of DNA is shown by this process. Alex Jeffreys has invented the process of DNA fingerprinting in 1985. Any biological samples such as blood, hair, saliva, semen can be used for DNA fingerprinting. DNA fingerprinting is also known as DNA profiling or molecular fingerprinting.
Molecular Markers
A known DNA sequence or gene sequence is present on a chromosome, and it is associated with a specific trait or character. It is mainly used as a genetic marker of the molecular marker. The first genetic map was done in a fruit fly, using genes as the first marker. In two categories, molecular markers are classified, classical marker and a DNA marker. A molecular marker is also known as a genetic marker.
DNA Sequencing
The most important feature of DNA (deoxyribonucleic acid) molecules are nucleotide sequences and the identification of genes and their activities. This the reason why scientists have been working to determine the sequences of pieces of DNA covered under the genomic field. The primary objective of the Human Genome Project was to determine the nucleotide sequence of the entire human nuclear genome. DNA sequencing selectively eliminates the introns leading to only exome sequencing that allows proteins coding.
![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.](/v2/_next/image?url=https%3A%2F%2Fcontent.bartleby.com%2Fqna-images%2Fquestion%2F0e3e796a-5332-4c60-9560-1058ec3b0add%2F8e08f05e-fa58-40d3-b9e8-789a7832b7b1%2F2a9omgz_processed.jpeg&w=3840&q=75)
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