Need help! Please explain.. thanks Interpretation Questions 3: Primer Design and

Question

Need help! Please explain.. thanks Interpretation Questions 3: Primer Design and Sequencing 1. Your advisor gives you the cloning plasmid pUCPIB2, containing the yeast gene PIB2. She wants you to move the PIB2 open reading frame (ORF) from pUCPIB2 into the expression vector pMCB473, so that you can express and purify the protein. A schematic diagram of the PIB2 ORF (in grey) in pUCPIB2 is shown below and includes a close-up of the first and last bases of the ORF. The expression vector pMCB473 is also shown below. (All arrows on both diagrams show the presence and direction of promoters). Ndel Pl pMCB473 pUCPIB2 Your advisor tells you to insert the gene at the Ps and Ndel sites in the MCS of the vector PstI 5'-C TGCA G-3 3-G ACGT C-5 NdeI :5-CA TA TG-3'cut sites 3-GT AT AC-5 In order to do this you must create these sites at the appropriate ends of the gene using PCR. Design the best forward and reverse primer that will allow you to amplify the entire PIB2 ORF and insert it into pMCB473 for subsequent expression of the entire protein. Forward primer Reverse primer Note the following in your design; 1) Your primers must allow you to produce a PCR product which when cloned into pMCB473 will allow you to express the entire protein. 2) In order to design the optimum pair of primers you should try to stick to as many of the rules and considerations of primer design discussed in PCR lectures 1 and 2 (the better your primers the more points you will get:)

Explanation / Answer

FORWARD PRIMER 5'- gtgcagTGAACTGCGTTGCATAGTGT- 3'

REVERSE PRIMER 5'- catatgTTGATGAGGTTAAGGTCTGTG-3'

Forward primer

length 20

GC% 45

Tm 55

Reverse primer

length 21

GC% 42

Tm 49

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