Already have an account? Get multiple benefits of using own account!
Login in your account..!
Remember me
Don't have an account? Create your account in less than a minutes,
Forgot password? how can I recover my password now!
Enter right registered email to receive password!
You have discovered a novel alternatively spliced gene (gene X) and you wish to study its regulation in an in vitro splicing assay. The alternatively spliced product retains intron 2 and is produced only in hepatocytes, whereas the standard mRNA is expressed in fibroblasts. The structure of the standard mRNA and the alternatively spliced form are shown below.
Conveniently, you have two cell lines available (one is derived from hepatocytes and the other from fibroblasts) that also show the same splicing patterns of this mRNA as that seen in tissue. You have already prepared a nuclear extract from each of these cell lines that you hope will provide the essential splicing proteins and snRNAs that are required for splicing your RNAs in an in vitro splicing reaction. You have already used PCR to amplify a genomic clone of the whole gene, which includes introns and exons. The primers are shown below with arrows. The 5' ends of the primers contain unique restriction enzyme sites to facilitate cloning in the later steps.
A. Briefly describe the major steps you would take to use the pcr product (500 bp) to generate a 32P-UTP labeled pre-mRNA run-off transcript (internal/uniformly labeled) that could be used in your in vitro splicing reactions. One or two sentences per step should be sufficient. Make sure you include details on the orientation of the PCR product in the construction.
B. You now prepare a freshly labeled gene X pre-mRNA from the construct you prepared from part a. You set up three in vitro splicing reactions in three different 0.5 ml tubes as described below. All have your labeled RNA but each contains a different protein extract or no extract. After incubation for 5 hours, which you have read should be long enough to get near complete splicing, you purify RNA from the reactions and run them on a denaturing PAGE followed by X-ray film exposure. The result is shown below. You suspect that the two darker bands (labeled a and b) may be the mature spliced mRNAs but you can't tell with certainty from this experiment. Using a technique we discussed in the lecture, suggest a follow-up experiment that could demonstrate that these two bands are in fact the correctly spliced mRNAs as depicted above. You can either use RNA purified from the gel shown below or you can go back and set up new reactions using your reagents. Explain briefly what you would do and what the experiment would show.
Discovery of Plant Hormones To date, five major classes of plant hormones have been discovered namely auxins, gibberellins, cytokinins, abscisic acid and ethylene. It is possi
The lithosphere contains all of the hard solid land of the earth crust and the semisolid land underneath the crust. The surface of the lithosphere is very uneven contains high moun
Q. What are the main bryophyte groups? The major bryophyte groups are the mosses, the hornworts and the liverworts.
What is Active site Active site : The site on the surface of an enzyme to which substrate or substrates bind.
Blockage of ducts In some bulls there is a blockage of the transport of sperm through the epidydimis usually caused by a congenital non-patency of some part of the vasa effere
Cleavage and Gastrulation Eventually, one spermatozoon fuses with the ovum to r restore the diploid genomic condition and activates all the potentials in the fertilized egg ce
Marine oils These oils typically contain large amounts of long - chain omega-3-polyunsaturated fatty acids, with up to six double bonds and they are usually rich in vitamins A
Q. Goals of prevention of diabetes mellitus? The goals of prevention are to delay the onset of DM and preventing or delaying the cardiovascular complications. Obesity should be
Define Needle selection for Suturing 1. Needle with a reverse cutting edge "the cutting edge is outside the curve" is preferable, because it prevents tearing, 2. Conventiona
GENETICS 1. The branch genetics started in 1900 after rediscovery of Mendel's work. 2. Gregor Johann Mendel is called Father of genetics. 3. The t
Get guaranteed satisfaction & time on delivery in every assignment order you paid with us! We ensure premium quality solution document along with free turntin report!
whatsapp: +91-977-207-8620
Phone: +91-977-207-8620
Email: [email protected]
All rights reserved! Copyrights ©2019-2020 ExpertsMind IT Educational Pvt Ltd