ComputerExerciseInstructionsf11

ComputerExerciseInstructionsf11 - Computer Exercise 1: Read...

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Computer Exercise 1: Read Designing in-frame protein fusion constructs (Mol. Exp. 4, p. M-47 of lab manual) and then carry out this exercise. The following handout goes through some of the steps that a researcher must carry out to determine which vector to use to construct an in-frame fusion of GST to their protein of interest. I am using the example of inserting GFP into pGEX, which is part of Molecular Experiment 4. Except for the final step (which you will carry out) I have provided all the information in text format to explain the process. However, if you are interested in learning how to do this yourself, I have listed a number of databases in the text and you can cut and paste the sequences below and carry out the other steps of the analysis yourself. But again you are only required to do the last step. Read through the steps and then complete the exercise at the end where you determine which expression vector to use. NOTE that I have posted both a pdf and MSWord version of this because one format can be easier to work with depending on your computer software- e.g in cutting and pasting sequences. If you open this in Word, see the note at the end about formatting sequences so that they line up correctly. If you are not able to cut and paste sequences you can write it out by hand. 1. Restriction Enzyme Choice The choice of restriction enzymes used in a specific experiment will depend on many factors, such as whether you already have your DNA of interest cloned in to a vector and what sites are available to isolate the fragment. One starts with a sequence of the starting plasmid or known DNA fragment. Enzyme prediction programs (e.g. http://tools.neb.com/NEBcutter2/index.php) are used to scan the sequence for the restriction sites and annotate them. For a simple exercise such as this one, you can also look up the enzyme recognition sequences (ie GAATTC for EcoR1) and search for it in a copy of the sequence in your word processing program. In our case, the GFP we start with is inserted into the MCS of pBS. GFP was already inserted into the MCS of pBS, and thus there are many sites in the MCS surrounding the GFP insert that could be used to isolate the insert coding for GFP. However, the sites used must compatible with the vector into which you are subcloning, pGEX in this case, and of course those sites cannot be present anywhere in the GFP insert or you will not isolate the entire GFP region. Using these and other considerations, see below), it was determined that EcoR1 and XhoI could be used to isolate the entire protein-coding fragment of GFP out of pBS-GFP AND clone it in the correct orientation in to pGEX. (Note that in cases where compatible enzyme sites are not available, PCR amplification can be used to amplify the gene of interest with primers that add on enzyme sites.) Below, I am showing the partial sequence of pBS-GFP plasmid with only the EcoRI (GAATTC) and XhoI (GAATTC) sites highlighted in bold (but parts of the MCS are underlined). In a
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ComputerExerciseInstructionsf11 - Computer Exercise 1: Read...

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