Isolation+of+Genomic+DNA


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Welcome to the Isolation of Genomic DNA Page!**

In order for our N2 normal, wild type worms to exhibit the dpy, or "dumpy", phenotype, we must preform RNA interference, or RNAi, on them. We must isolate genomic DNA from the N2 wild type worms by following the procedure below then amplifying a portion of the dpy-10 sequence, or gene of interest, via PCR. After we isolate and amplify the portion of dpy-10 gene, we must insert it into the L4440 plasmid which is used to transform the HT115 cells, the feeding strain of //E. coli// for the worms. HT115 cells make T7 RNA polymerase which binds to T7 promoters on the plasmid to transcribe the dpy-10 gene, creating double stranded RNA (dsRNA) which starts the RNAi mechanism.

// General Procedure: //
 * 1) Prepare worm lysis buffer by mixing 5ul of porteinase K with 95ul 1xPCR buffer (dilute 2x PCR buffer with dilution buffer)
 * 2) Place 3-4 medium sized worms in PCR tubes with worm lysis buffer. Make sure the worms are still alive by checking to see if the worms writhe in the lysis buffer.
 * 3) Centrifuge the worms to the bottom of the tube.
 * 4) Freeze the worms in the freezer (-20 degrees celsius) for 90 minutes. Freezing and thawing bursts the worms open.
 * 5) Thaw the worms by placing the tubes in a 65 degree Celsius water bath for 90 minutes.
 * 6) Denature the Proteinase K by placing the tubes in a 95 degree Celsius water bath for 15 minutes.







//Edited by Deo Deiparine//