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​​​​​​​Describe how you can use PCR to obtain a gene fragment X that can be cloned...

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Biology

​​​​​​​Describe how youcan use PCR to obtain a gene fragment X that can be cloned into aplasmid vector for expression of the recombinant protein. Includein your description how the PCR reaction will be performed, how theamplicon will be analyzed, the use of restriction enzymes, howcloning will be achieved, how you will know that you have arecombinant containing the insert gene fragment X and how you willdetect the recombinant protein.

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Designing primers for PCR based cloningThe basic PCR primers for molecular cloning consist ofLeader Sequence Extra base pairs on the 5 endof the primer assist with restriction enzyme digestion usually36bpRestriction Site Your chosen restriction sitefor cloning usually 68bpHybridization Sequence The region of theprimer that binds to the sequence to be amplified usually1821bpWhen selecting restriction sites you should use a DNA analysistool such as Addgenes Sequence Analyzer to allow you to identifywhich restriction sites are present in a given sequence You wantto choose enzymes thatDo not cut within your insertAre in the desired location in your recipient plasmid usuallyin the Multiple Cloning Site MCS but do not cut elsewhere onthe plasmidBonus It is helpful to choose restriction enzymes that can bothfunction in the same buffer as this will save time laterIn our example we will use EcoRI and NotI to ligate our cDNAinto the recipient plasmid Remember to insert your DNA in thecorrect orientation in the recipient plasmid by viewing the MCS andfusing the upstream restriction site to the forward primer and thedownstream restriction site to the reverse primerNext we need to examine the DNA sequence that we want toamplify and design primers that will bind to and replicate it Thefollowing image shows the ends of the ORF and how these are usedfor primer designBecause we are cloning an ORF we want to clone from the startcodon ATG to the stop codon TGA in this example Assuming youare amplifying from plasmid DNA rather than from genomic DNA or acDNA library roughly 1821bp is usually sufficient to givespecificity and to also be compatible with a standard PCR reactionTherefore our Forward Primer will    See Answer
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