To design the PCR primers, you must collect a lot of sequence information and analyze it. Some of the steps I will tell you about, others you will have to figure out for yourself. The gene names for these five genes are IDH1, IDH2, IDP1, IDP2 and IDP3. Below are some key steps to take and some web sites to consider. However, you might find other sites you prefer. No matter which web site you use, be sure to cite each one on your web pages. See the full list of expectations for web pages here.
First, you will need to find all five DNA sequences. Start at this URL or you might prefer this one (Genebank).
Next, you will need to get restriction maps for each gene. Try here. Be sure to note the length in bp of the ORF.
Now you will need to design the PCR primers. (Helpful Hints Here) You want to keep the reading frames in mind. Each PCR primer should be about 20 bases long. However, more important than length is melting temperature (Tm). We would like all 10 primers to have about the same Tm so use this web page to help you calculate approximate Tm.
Finally, write out the DNA and amino acid sequences for the fusion proteins from His6 tag through the first 10 amino acids of each IDH.
Map of expression vector from Qiagen (pQE 30 UA).
PCR Primers used for this lab.
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