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.
Why is drosophila a convenient animal for the study of linked genes? The fruit fly drosophila is appropriate for the study of Genetics because it presents many distinct traits
LYMPH - Lymph is a mobile connective tissue comprising lymph plasma (fluid) and lymphcorpuscles (cells). COMPOSITIO N - (i ) Lymph Plasma - It is simil
Phases of Respiration in Animals The procedures of respiration in these animals have the following phases. i) External respiration is usually described as breathing. It inv
Explain about Battery Battery: Two electrodes (zinc and carbon) placed in electrolyte (dilute sulfuric acid). Chemical reactions in electrolyte force positive charges to
Explain the Scaling from individuals to Ecosystems? Models that explain how individual organisms acquire energy and materials, and how they use them for survival, growth and re
General characteristics of phylum
Effect of processing on mineral content of foods Minerals are comparatively stable under processing conditions such as heat, light, use of oxidizing agents and extremes in pH.
draw a well labled diagram of plasmotomy
Hydra – Metazoa The animals of phyla Cnidaria and Ctenophora are made up of only two germ layers: ectoderm which forms the outer covering of the animal and endoderm which is t
Composition of Plant and Animal Tissues Food supplies nutrients popularly known as major and minor nutrients based on the quantities in which they are required for the living b
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