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Topic: RNA extraction and PCR cloning

Your results and comments should be contained within the space provided in this section of the course handbook and should be your own work; it is understood that you have worked in pairs and will have discussed your answers with your partner, but your answers must be expressed in your own words. If you want to word-process your answers to the longer “Introduction” and “Discussion” sections please use 12 pt type and paste them into the space provided. Attempt to answer all of the questions and give sufficient information that the reader can follow exactly what you have done (e.g. state quantities and gel percentages wherever these are not fixed in the appropriate Procedure). Brevity, clarity and accuracy will be rewarded. Any sample borrowed from another group should be indicated in the report giving the name of the other group. 5% of the marks will be awarded for presentation.
INTRODUCTION (25%)                Max. 400 words
Describe what you hoped to achieve (the aims and rationale of the experiments), providing any relevant background information.

Results (25%)

What is the general purpose of LiCl used in the RNA preparation? State which other method could have been used?


How does the relative amount of visible RNA on your RNA gel compare with your spectophotometry data.


At which stage of the practical were the Zn finger motifs selected. Explain how Zn fingers are isolated from the rest of the RNA species.


How do you account for the banding pattern observed in the PCR reaction


What do the “no reverse transcriptase enzyme” and “no RNA” controls tell you?


Which purification method would give a cleaner PCR product for subsequent ligation and why?


Discuss the results you obtained in the ligation reaction. What do the various controls tell you?


Draw a restriction map to illustrate how the size of inserts from recombinant clones cut with PvuII relate to the size of purified PCR products put into the ligation reaction. To do this you will need to take account of the position of the PvuII sites in the pGEM-T Easy vector (see Figure 5 and Introduction to P5). Were any of your clones cut into more than 2 fragments with PvuII? Why might this happen?


Draw a map to show how the sequence relates to the insert in the recombinant plasmid (i.e. show the orientation of the insert relative to the two sequencing primers). To do this you need to know that the forward and reverse sequencing primers are complementary to nucleotides 2959-2975 and 176-192, respectively, in the pGEM-T Easy vector shown in Figure 5). Indicate which primer you used.


Giving the top ten (approx.) sequence matches that were retrieved, comment on the nature of any protein encoded by the recombinant clone sequenced?


Draw a flow diagram showing the different steps of your samples from P1 to P7. This should include the different sample characterisation experiments as well as the amounts or concentration of samples. (5%)

DISCUSSION (35%)            Max. 600 words

i) briefly summarise your main findings; ii) focussing on a problem you encountered, identify one or more reasons the experiment may have failed and suggest improvements that would eliminate the problem if the experiment was to be repeated; iii)   speculate on the significance of your main findings and suggest ways in which the experiments might be continued.



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