• May 27th, 2015

Molecular Cloning

Paper, Order, or Assignment Requirements

 

 

** you will find in attachment file that describe the topic of report.
** 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 

** please avoid plagiarism
** each part on two and half page 
** font must be 12 
** the results have been attached some as images and some as graphs so, try to put all of them in the results section and explain them them

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