Home
Editor: Katie Cavanagh

Picture_13.png

The isolation of the genomic DNA from the worms is the first major step in the RNAi experiment. First, it is important to recognize that there is a difference between genomic DNA and a plasmid. The genomic DNA that is being extracted from the worms is the complete set of genetic information found in the chromosomes of the worms that codes for all genes present in the worms. This DNA differs from that of a plasmid because a plasmid is a circular molecule of DNA that replicates independently and tends to naturally occur in bacteria such as the feeding strain of E. Coli which we are using. Genomic DNA that has been cut into a linear piece can be inserted into a plasmid as occurs in our experiment. (Refer to Diagram 1)

Diagram 1: Genomic DNA versus Plasmids

14_1.jpg
From: http://www.biologie.uni-hamburg.de/b-online/library/onlinebio/BioBookDiversity_2.html

Before the desired gene can be silenced it must first be isolated. Initially the worms must be grown according to the process described in the Worm Care section of the wiki. The following procedure lyses the worms by first freezing them so that they swell and then heating them so that the worms will burst open and there DNA will no longer be contained within them. The Lysis Buffer with Proteinase K into which the worms are placed aids this process. The buffer, as its name suggests, helps with the opening and lysing of the worms. The enzyme Proteinase K in the buffer breaks down the proteins in the worm as the DNA is released. However, it is not necessary to isolate the DNA from the worm debris since the next process, PCR, only amplifies the DNA since a specific sequence will be targeted. With this recovered DNA, PCR, a process that makes copies of specific sequences of DNA as determined by specific primers, can be performed on the DNA and thus the desired gene, Rol6, can be amplified and inserted into the L4440 plasmid that will ultimately be fed to the worms. This step of extracting the DNA is imperative as placing the gene in the plasmid in the location we do enables the silencing of the Rol6 gene.


Materials/Tools

PCR Tubes
3-5 Worms
Lysis Buffer with Proteinase K
Worm Pick
Dry Ice or a Freezer at
4ºC
Thermal cyler
Waterbath


Procedure

NOTE: Before beginning, create a 1X lysis buffer by diluting 10 microliters of 10X lysis buffer with 90 microliters of deionized water. Next, add 95 microliters of the 1X lysis buffer created to 5 microliters of Proteinase K to create the lysis buffer with Proteinase K that will be used in the procedure.

KC_Final.png

  1. Using a worm pick, place three to five worms into a PCR tube containing 10 microliters of Lysis Buffer with Proteinase K. (Ensure that worms are moving in the tube to show that they are still alive.)
  2. If dry ice is available, freeze the tube on drive ice for 10 minutes. If dry ice is not available, the PCR tube can be chilled in a freezer at 4ºC for 1 hour.
  3. Heat the tubes in the water bath at 65ºC for 60 to 90 minutes.
  4. Heat the tubes in the thermal cycler at 95ºC for an additional 15 minutes.
  5. Store the tubes at 4ºC until PCR is performed to amplify the now extracted genomic DNA.
Katie's_Flowchart.png