Molecular Cloning of a gene. You have been given these genes. Find the restriction enzyme you will use that would not cleave your gene. But assist in inserting into DNA of an organism. 1) Find Forward Primers and leave at least few nucleotides at the start to allow the Polymerase enzyme to attach itself to the gene to glue on the primers For example, on the example i have uploaded… "5’atacCTCGAGatgacccggctgc 3’" Capital Letters are the foward primers. The letters before are extra nucleotide that allow the enzyme to be seated. Also include Tm (you can use 3rd party website to determine Tm) and Do the same with the reverse primers, such as figuring which kind of restriction enzyme to use and primer and use 3rd party website to determine complimentary base pairs. and figure out the Tm. The forward and reverse Tm should not be no more than 5 degree Celsius different. If so replace or remove any GC—>AT bonds that are added to the beginning of the gene that allows the polymerase to sit on (since it would be junk regardless) Then figure out the Annealing Temperature.
Last Thing to do is to sketch/draw the gene structure such as the picture example is uploaded. You can either hand write it to make it easy for you or use software to upload it. The 4 charts are basicly the 4 pictures(since drawing the gene is a lot of work. MARK GENE, AMP(ampicillin resistant) , RESTRICTION SITES. Also Find PCR program which is widely available online. Lastly if you can find the GFP gene that would be nice too! Its one of the 4 genes. The Docx i uploaded give you the directions too.
