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Subcloning & Yeast two-hybrid

This should convey the point of the mini-project in 15 words or less.

**Introduction (about 200 words):
You can use figures in addition to text in this section if you like.

• Subcloning
-What are plasmids and how can they be used?
-How can a piece of DNA encoding a protein of interest -be isolated from a
-How can such a DNA fragment be introduced into another plasmid?

• Yeast two-hybrid
-What techniques can be used to verify that subcloning has successfully
produced the desired plasmid?
-What are the main applications of this technique?
-How does it work?

• Finish the Introduction with a very clear statement of the AIM of the Project

**Materials and Methods:
Include a flow chart of the techniques used
Do NOT write out what you did – just a summary to demonstrate that you understand the aims
of each prac & how they linked together to form the project.
 Steps only
 No details of reagents etc.

**Results (about 300 words plus figures):
-Present and describe your individual results and the compiled class data.
*You will use the notes you made during the practical classes, plus photos of gels and yeast plates, and class transformation/streaking data.
– Include in your report figures showing your gels and plates your samples (and what they were) and label the molecular weight markers with the size of each fragment (in nucleotides or kilobases).
*Must be labelled correctly and be described in text.

In the text of this section, make sure you address the following points:
• What size fragments did you expect from the restriction digestion of each plasmid in
practical 1? Based on your results, which plasmid did you cut?
• What PCR and restriction digests patterns did you expect in practicals 2 and 3, if the
colony you picked contained the desired Bad-pGBT9 plasmid? Do your results match
these expectations?
• Which plasmid(s) did you transform into yeast in practical 7? Did any transformant
colonies grow on the -LW plate? Considering the entire class data, which
transformations generally yielded colonies on the -LW plates? Explain these results.
• Which transformants grew on your -LWH plate in practical 9? Did this outcome match
the whole-class data?

**Conclusion and discussion (about 400 words)
• Re-state your Aim and describe the extent to which it was achieved.
• Outline the purpose of including the “negative control” PCR reaction and culture in
practical 5. Did these behave as expected? If so, what can you conclude the results from the PCR and miniprep from the tubes inoculated with the bacterial colony. If the controls did not yield the expected results, why not?
• Did the rest of your results match your expectations? If you had any surprising results, suggest possible reasons for these. Was it human error (eg did you not pipette the correct volumes, or threw out the supernatant when you were meant to keep it)? Could defective reagents or equipment be responsible, such as enzymes that had lost activity, PCR machine/heatblock not achieving specified temperature? (Consider class data in relation to this possibility).
• Write an overall conclusion describing very briefly what the aim of the project was, what the findings were and how it can be improved in the future.

at least six different references,
In-text referencing should occur in the Introduction,
Materials and Methods and Discussion.
Font: Times New Roman
Size: 12 point
Lines: Double spaces

In-text referencing should occur in the Introduction,
Materials and Methods and Discussion with the pg no.

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