Molecular cloning

this report consist of 2 part
– part A: Molecular cloning of GFP gene into pBCSK vector
– part B Heterologous GFP protein expression in E.coli.

** please follow these structures to write the report for two part
and each part must to be 2 pages and half

# structure of part A: Molecular cloning of GFP gene into pBCSK vector
1_ Introduction
-Background

2_ Aim of experiment
-include your understanding of the techniques used –molecular cloning, transformation, GFP as a visual reporter
-Give a real life example where these techniques are used

3_ Results

Restriction digestion of pGFP and pBCSK
– Show your gel’s image
– Explain your bands
– Identify location of GFP, pGFP vector backbone and pBCSK vector backbone
– Include sizes in marker

Show E.coli colonies containing recombinant pBCSK-GFP plasmids.
– Show GFP fluorescence of the E.coli colonies

4_ Discussion
– Discuss the results. What did you find?
– Were the results as expected? Why? Why not?

5_ References
– Minimum of 2 references
– Use Vancouver referencing style.

# structure of part B: Heterologous GFP protein expression in E.coli.

1_ Introduction
– Background

2_ Aim of experiment
– include your understanding of the techniques used –Bradford, SDS-PAGE, Western blotting
– Give a real life example where these techniques are used

3_ Results
Protein Bradford assay
– Include raw data as table and standard curve as fig.
– Label tables and figures correctly
– Write out protein concentration for each sample and how much of original protein you added to make 100 uL of SDS-PAGE – 1 mark ug/mL to run on the gel

SDS-PAGE gel (stained)
– Include instant/coomassie stained SDS-PAGE gel
– Label figure with your samples – identifying samples in each lane
– Identify location of GFP
– No discussion here – only report on the results you observe
– Include sizes in marker

Western blot
– Fig of western blot
– Include sample ID and sizes of band in the marker
– GFP = 31 kDa
– Identify location of band

4_ Discussion
– Discuss the results. What did you find?
– Were the results as expected? Why? Why not?

5_ References
– Minimum of 2 references
– Use Vancouver referencing style