Report 1: Molecular Cloning Exercise – Instructions
Learning Objectives
Once you complete this report you should be able to:
Read and analyse a plasmid map
Calculate the sizes of DNA fragments that result from restriction endonuclease digestion and PCR
Predict the positions, after agarose gel electrophoresis, of DNA fragments that result from restriction endonuclease digestion and PCR
Predict the amino acid sequence of an encoded protein.
Background
Imagine that you have recently joined a research laboratory and have been asked to clone the coding sequence of a novel protein (RKSG) into a plasmid vector to allow expression as a GST-fusion protein, very similar to the GST-GFP construct used in the LEAPS practicals and the same principle as the His-tagged Ta1a construct used in ALURE.
Your supervisor provides you with a protocol for the PCR amplification of the coding region of the new protein, including the sequence of the primers RKSG-Fwd and RKSG-Rev. Your supervisor asks you to amplify the coding region following this protocol.
You then have to clone the amplified fragment into the GST-containing expression vector pGEX6P2 (related to the pGEX2T vector used in LEAPS) and determine if the cloning has been successful.
To accelerate the cloning process the decision has been made to cut the vector with Sma I, giving blunt ends, and then ligate the blunt-ended PCR product into the vector.
Information Provided
A restriction map of the plasmid vector pGEX6P2
Details of the multiple cloning site of the pGEX6P2 plasmid
DNA sequence of the region containing the coding sequence of RKSG
The sequences of the primer pair RKSG-Fwd and RKSG-Rev
The sequences of the generic screening primers – SCREEN-F and SCREEN-R. Note that these primers bind to positions in pGEX6P2 some distance from the multiple-cloning site and so you won’t find their binding sites in the MCS diagram.
The recognition site and cutting patterns of key restriction endonucleases
The genetic code
A template gel image
What you need to do.
You need to create a document that includes the information required for both Task 1 and Task 2.
Assignment submission
Please submit your assignment online (through Turnitin). We expect that a lot of this assignment will include hand drawings – hand drawings are still the easiest way to show working for the skills we are testing here. To submit your hand drawing through Turnitin, just take a photograph of your work and import it to a Word document as a PDF or JPEG. Sometimes Turnitin refuses to accept assignments with a lot of PDF or JPEG material, so just type a few lines of text above the first PDF/JPEG in your assignment. Your name, student ID, and the name of the assignment will do. Once you do this, Turnitin will recognise the text and accept the assignment.
The Assignment Tasks (2 tasks total, 30 marks total)
