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.
what are the ecological importance of molluscus
Minerals and reproduction The mechanism of mineral-reproduction interactions is not fully understood because of the complexity of neuro-hormonal dialogue. Some minerals act direc
Describe the process of excitation-contraction coupling (ECC) in the heart. How do adrenaline, noradrenaline and acetylcholine alter heart rate and or force?
Closed Mitral Valvotomy : This has been 1-eplaced by balloon mitral valvotomy (BMV) in most centres. It is done in cases of either pure mitral stenosis or with .triv
Define General procedure of titration - Nutritional Biochemistry? In general a titration requires that we add precise amounts of the known titrant to the unknown analyte or ti
what are peptides and poly peptides hormones
Obesity a health hazard: The factors of obesity are complex. Obesity is due to over eating and excess of energy intake. The obese child when grows up will be a target
Introduction: There are different technologies available for dealing with waste water management and excreta. Some are suitable for dealing off-site sanitation and other can b
1. Describe how the various molecular mechanisms act to regulate transcription of the genes within the lactose operon of E. coli . 2. Transcription of a eukaryotic protein-codi
Intracellular Digestion We all know that unicellular organisms do not have a separate alimentary canal system. All the functions of life are carried out inside a single cell.
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