Q.7a) You are seeking to clone a gene into pEGFP-C1 (see plasmid MCS map in Q.4). Which of thefollowing forward primers with added RE sites (in capitals) will result in your gene being in-framewith EGFP? The start codon is underlined and the RE being used is shown in brackets.(i) 5'-nnCTCGAGatggacgatgtagcacagctt-3' (Xhol)(ii) 5'-nnGGATCCatggacgatgtagcacagctt-3' (BamHI)(iii) 5'-nnGAATTCatggacgatgtagcacagctt-3' (EcoRI)b) For the primers that will result in the gene being out-of-frame with EGFP, make alterations to theprimer sequences to correct the frame.

To determine which primers will result in gene being in-frame with EGFP , we need to analyze…

Answered: Q.7 a) You are seeking to clone a gene…

Human Anatomy & Physiology (11th Edition)

11th Edition

ISBN:9780134580999

Author:Elaine N. Marieb, Katja N. Hoehn

Publisher:Elaine N. Marieb, Katja N. Hoehn

Chapter1: The Human Body: An Orientation

Section: Chapter Questions

Problem 1RQ: The correct sequence of levels forming the structural hierarchy is A. (a) organ, organ system,...

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In mammalian cells, genes that are expressed in a particular cell are reported to undergo replication during the first half of S phase, and genes not ex- pressed in that cell are replicated in the latter half of S phase. Briefly describe an experiment that could lead to this conclusion. You might consider ap- proaches that involve 5-bromouracil incorporation.
explain please: Spike-Driven Syncytia Formation Is Coupled to S20 Fragment Generation in the Presence of ACE2. To investigate the functional and biochemical signatures of spike (S) protein upon engaging its receptor ACE2 in live cells, we first utilized a cell–cell fusion assay to obtain potentially cleaved spike protein products from syncytia . In this system, HEK293T cells transfected with plasmids encoding WT spike readily formed syncytia after coculturing with HEK293T cells expressing human and Vero E6-ACE2, as well as colorectal adenocarcinoma Caco-2 and lung adenocarcinoma Calu-3 cells expressing endogenous ACE2. We then collected these adherent syncytia and immunoblotted for cleaved spike species using a rabbit polyclonal antibody specifically detecting the S2 ectodomain through standard reducing Tris-glycine sodium dodecyl sulfate polyacrylamide gel electrophoresis. This experiment revealed that spike expression in HEK293T cells displayed full-length S and autocleaved S2…
Table 1 shows a list of restriction endonucleases with their recognition sequence and the sites of cleavage indicated by arrows. Table 1 Enzyme name Recognition sequence and position of cut 5'GIAATTC3 5'G!GATCC3' 5'GIGTACC3 5'GCIGGCCGC3' 5'IGATC3' 5'GGTACIC3' 5'ALGATCT3 EcoRI ВатHI Аcс651 Notl Sau3A Kpnl BglII (i) Which restriction enzyme(s) produce blunt ends? (ii) Are there any pair of neoschizomers in the list? Explain. (iii) Are there any pair of isocaudomers in the list? Explain.
Suppose you have a 1-kb segment of cloned DNA that is suspected to contain a eukaryotic promoter including a TATA box, a CAT box, and an upstream GC-rich sequence. The clone also contains a gene whose transcript is readily detectable. Your laboratory supervisor asks you to outline an experiment. (1) determine if eukaryotic transcription factors (TF) bind to the fragment and, if so, (2) identify where on the fragment the transcription factors bind. All necessary reagents, equipment, and experimental know-how are available in the laboratory. Complete the outline of an experiment, which determines if eukaryotic transcription factors (TF) bind to the fragment. Assume all necessary reagents, equipment, and experimental how are available in the laboratory. Drag the terms on the left to the appropriate blanks on the right to complete the sentences. Not all terms will be used. For the band shift assay, two samples of the DNA fragment are analyzed by agarose gel electrophoresis: one sample is…
When separating proteins from cellular extractions, what electrophoresis method works best and what protein characteristics need to be taken into account that are not a problem for nucleic acid-based molecule? Describe the process and in your answer.
Qiii and Qiv only
If an enhancer is placed on one plasmid and its corresponding promoter is placed on a second plasmid that is catenated (linked) with the first, initiation is almost as effi cient as when the enhancer and promoter are on the same plasmid. However, initiation does not occur when the two plasmids are unlinked. Explain.
Which of the two restriction sites in pQE vector will be appropriate to allow full length insertion of your gene? These restriction sites will serve as linkers on both ends of your gene
For the experiment shown Fig. 6.30 and 6.31, what is the advantage to use a labeled incoming nucleotide (α-32P-UTP or α-32P-CTP), not labeled reagent 1 itself? Please draw a diagram to help explain your answer by contrasting the RNA polymerase labeled with α-32P-Reagent (Panel A) vs α-32P-UTP (Panel B).
in ecoli, a ligase was deleted from the genome. the cells were then placed in a slighly higher temp. would the ligase deletion matter?
E17. Gene mutagenesis is also used to explore the structure and function of proteins. For example, changes can be made to the coding sequence of a gene to determine how alterations in the amino acid sequence affect the function of a protein. Let's suppose that you are interested in the functional importance of a particular glutamic acid (an amino acid) within a protein you are studying. By site-directed mutagenesis, you make mutant proteins in which this glutamic acid codon has been changed to other codons. You then test the encoded mutant proteins for functionality. The results are as follows: Functionality (%) Normal protein 100 Mutant proteins containing Тугosine Phenylalanine 3 Aspartic acid 94 Glycine From these results, what would you conclude about the functional significance of this glutamic acid within the protein?
Is RNA polymerase saturated with substrates in vivo? Describe experiments that might indicate whether RNA polymerase is operating at Vmax with respect to its nucleotide substrates.

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