Biochemistry 2280A Lecture Notes - Cloning, Plasmid, Chain Termination
Document Summary
Dna ligase will recongnize the nics and use energy of atp. Ligase has harder time getting blunt ends together. It is like glue that puts dna back together. Ligase requires phosphates at the end of the dna. Steps in cloning: digest gene with restrictive enzyme. That will release our gene from the pcr product. Result in recombinant molecule: we need to get this into the ecoli- this process is called transformation. Chain termination sequencing involved the enzymatic synthesis of a dna strand in the presense of base specific. It lacks 2" and 3" oh. (we only care about the 3" oh)- this causes it to be defective so the sequencing stops. We need dna primers, dna to be sequences, dna polymerase, ddntp (small amount), dntp (nucleotides).