You are interested in synthesizing a variant of Synechocystis sp. pcc 7803 ferredoxin having mutation (E) at its 38th residue. The gene is encoded in a pET21b vector (5.3 kbp). Estimate the amount of time needed to run a PCR for the site-directed mutagenesis for 25 vs 30 cycles.
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You are interested in synthesizing a variant of Synechocystis sp. pcc 7803 ferredoxin having mutation (E) at its 38th residue. The gene is encoded in a pET21b vector (5.3 kbp). Estimate the amount of time needed to run a PCR for the site-directed mutagenesis for
25 vs 30 cycles.
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- the forward primer used in this experiment incorporates part of the cell recognition site, GGCC. How is this different from the sequence of the human TAS2R38 gene? What characteristic of this PCR reaction allows the primer sequence to override the natural gene sequence? Draw a diagram to support your contention.Bacteriophage lambda is as one of the routinely used molecular cloning vectors, which alsoserved as a model system for the study of bacteriophage morphogenesis, DNA replication, andgene regulation.a) With the aid of a diagram, generally narrate how a foreign gene can be inserted into alambda insertion vector and subsequently infect an Escherichia coli cell.b) You are cloning a 7.5 kb gene into a lambda gt10 vector, utilizing a restriction site whichspecifically present in the vector. State the restriction site that you can use for this purposeand suggest a screening procedure to indicate successful integration of the gene into thehost genome.What are the key modifications to the PCR primers, SHFw1 and SHRv1 in order for you to clone the PCR amplified Sonic Hedgehog into the multiple cloning site (MCS) of pSELECT-CGFP-blasti. Explain your answer in detail
- The sequence at one end of one strand of the Drosophilatransposon Mariner is shown below (dots indicatesequences within the transposon):5′ TTAGTTTGGCAAATATCTCCCTTCCGCCTTTTTGATCTTATGT... 3′You obtain a mutant bacterial strain tagged with anengineered Mariner transposon, cut the genomicDNA from this strain with the restriction enzymeMboI (whose recognition site is ^GATC), and circularize the resultant DNA fragments by diluting therestriction enzyme digest and adding DNA ligase.a. Design two 17 bp PCR primers that you could useto identify (by inverse PCR) the gene into whichthe transposon inserted.b. What DNA sequence will be amplified from thecircularized fragments of the mutant genome?Show the extent of this DNA sequence on a mapof the genome of the mutant strain, indicating thelocations of the transposon insertion and any relevant sites for the enzyme MboI.After Drosophila DNA has been treated with a restrictionenzyme, the fragments are inserted into plasmids and selected as clones in E. coli. With the use of this “shotgun”technique, every DNA sequence of Drosophila in a librarycan be recovered.a. How would you identify a clone that contains DNAencoding the protein actin, whose amino acid sequenceis known?b. How would you identify a clone encoding a specifictRNAThe restriction endonuclease NotI recognizes the octanucleotide sequence GCGGCCGC. Calculate the expected number of NotI cleavage sites in the bacteriophage λgenome, a linear DNA duplex 48.5 kbp in length with a (G + C) content of 50%.
- The DNA sequence of one strand of a gene from threeindependently isolated mutants is given here (5′ endsare at left). Using this information, what is the sequence of the wild-type gene in this region?mutant 1 ACCGTAATCGACTGGTAAACTTTGCGCGmutant 2 ACCGTAGTCGACCGGTAAACTTTGCGCGmutant 3 ACCGTAGTCGACTGGTTAACTTTGCGCGWhat would three possible reasons be that a PCR wouldn't work after cloning a TAQ sequence into a His-tag vector making three different constructs and verifying via SDS-PAGE that the TAQ protein is there?You are amplifying both gst::egfp mRNA and 23S rRNA in the PCR steps. These are two different types of RNA (messenger and ribosomal, respectively). Could an E. coli mRNA rather than an rRNA be amplified as a reference gene? Why? What are the main criteria for selecting a reference gene?
- E. coli strain BW25113 can grow on (D) Arg as the sole carbon source. A scientist working with you on this project has discovered that mice encode a protease that cancleave between (D)Arg‐(D)Arg. a. In a set of discrete steps outline how to construct this genomic library so that recombinant expression can be performed in E. coli b. Once the genomic library has been constructed, describe in DISCRETE steps how you would isolate the DNA sequence that encodes for the protease that cleaves between (D)Ag-(D)ArgRecall that constructs used for floxing a gene contain,within one of the gene’s introns, two loxP sites flanking a gene for neomycin resistance (Fig. 18.11a). AloxP site is only 34 base pairs long, as shown in thefollowing figure.ATAACTTCGTATA ATGTATGC TATACGAAGTTATInverted repeat Spacer Inverted repeatExplain how you could use PCR to generate a neomycin resistance gene flanked by loxP sites, starting witha plasmid containing a neorgene. If you had the intronof the target gene cloned in a plasmid vector, howcould you insert your PCR product into the intron?Design a site directed mutagenesis experiment to convert the wild type GFP sequence into the S65T mutant. You must include the sequence of the primers, and outline of the PCR conditions you would use, and the protocols for the mutagenesis and transformation of bacteria with your plasmid containing the mutation.