I pray that I’m back, and so I get to scream like george costanza, “I’m back baby!”
this, my lemon drops, is a great example of robust protein priming…the top bands are at least. what you see is the beta particles emitted from the alpha P32 labeled dATP, my radioactive label. it gets incorporated into the newly synthesized cDNA via the reverse transcriptase. the protein covalently attached to the full length cDNA product is about 120 kDa in size; that’s the top band. the bottom smear if you will, is snapped-back RNA primed cDNA products, with labeled dA incorporated. the RNA template folds onto itself, forming a secondary structure and it acts as it’s own primer.
you see, the polymerase that I study is one of a kind. it selectivity for DNA synthesis initiation is extremely flexible, yet has it’s limits. it’s the only polymerase with the ability to use RNA, DNA or protein to prime DNA synthesis. just in this gel, you see two example of DNA synthesis initiation, and the final products (protein and RNA used as primers).
I do this before I use my partially purified RT in other significant experiments, to gauge activity and equalize how much to use per reaction for further experiments; hence, this is a check of activity, with and without RNA template (the negative control are the lanes with no template, designating that no product was synthesis, as there shouldn’t be any to begin with).