Results for AGE

1. Cloning DNA in bacteria and finding the ligand binding factor for NHR-85a Christopher Place & Gordon J. Lithgow The Buck Institute for Research on Aging, 8001 Redwood Blvd; Novato, CA 94945 USA Abstract NHRs(Nuclear Hormone Receptors) are ligand binding factors that effect the transcription process of DNA to RNA. There are 284 NHRswithin a Caenorhabditiselegan, each one with its own distinct function, the average human has about 48. The task handed to me, was to isolate and clone NHR 85a by tagging it to a Pbind plasmid. Once obtained, we can put NHR-85a into a lipid extract or do a chemical library on the NHR and then check for any luciferase activity, which tells us a ligand has bound to our NHR. And from there we can use the ligand, whatever it may be, and add it to the C. elegans and see what the NHR promotes. Transformation -A way of cloning our desired plasmid into DH5a strain bacteria Agarose Gel Electrophoresis (AGE)- -An agarose based gel that we insert our DNA fragments into, then apply an electrical charge, our DNA is separated by size, giving us the approximate band size Digestion- -A way of cutting our plasmid at certain areas to make it linear for AGE Polymerase Chain Reaction (PCR)- -A way of amplifying existing DNA so that we can study it further Ligation- -Similar to digestion, but, we add restriction enzymes to our DNA so that they attach to the ends of the DNA so that our DNA will be inserted into a plasmid 5’-3’ Mini-Preps- -Small kits essentially made for extracting DNA in small quantities NanoDrop- -A fascinating contraption that takes about 1ul of sample and can give you the almost exact concentration Western Blot- -How we determine the concentration of a protein sample or to see if our desired sample is even there Results for AGE 1 Kbp 1 Kbp 64.1 64.2 64.3 64.4 64.5 xho1 xho1 xho1 xho1 xho1 M 80 8 64 85a 14 M pbindb/xpbinds/x 8 48 Ran at 110v for 50 minutes Gel after our ligation, ran simply to make sure our DNA was right Ran at 110v for 50 minutes After our restriction digestion, ran to assure us of correct size Introduction What are C. elegans? -Caneorhabditiselegans, are nematodes, approxomatly 1 mm in length that feed on bacteria -About one out of every 10,000 C. elegans are male, the rest are hermaphrodites (about .01% are males) Why research them? -C. elegans have short lifespans, perfect for testing long term effects of drugs -Simple organisms with simple mechanisms, easy to maintain -A good amount of their genes are homologues to humans Nuclear Hormone Receptors- -NHR’s are ligand-activated transcription factors that regulate a specific gene expression Pbind plasmid- -Plasmids are circular free-floating DNA found in bacteria - Pbind is a manmade plasmid with preset fragments that have desired traits -We attach the NHR to the plasmid in order to transport the DNA to mammalian cells Western results 1 Kbp N2 worms RNA PCR cDNA Primers Final test to make sure our cloned DNA was actually NHR-85 Digestion Restriction Digestion AGE gel Gel extraction Quantify Conclusion NHR-85a still has an unknown function Human error is always a variable Insufficient time to come up with actual data Future direction Extend the duration of the internship Have the plasmids already made beforehand Improve upon minor errors made Bacterial Colonies Ligation Transformation Mini-Preps Party Digestion AGE gel DNA is viable No Transfer to mammalian cells Quantify Yes Acknowledgements Lithgow lab Western blot lipid extract Collect cells