Isolating+Bli-1

// Isolation of the Genomic DNA //

//**What is Genomic DNA?**// The Genomic DNA differs from the plasmid contained in the cell. In this lab we are isolating the chromosomes in the worm //(C. elegans//). The chromosomes represent all of the genetic information the worm contains. A plasmid, however, is simply a circular section of DNA. This can occur in cells but is not what we are after; the plasmid only denotes a part of DNA. The image below represents the different between the chromosomal DNA and a plasmid. Although it is not a great representation of the actual DNA, one can see the difference between the two. In this lab we were trying to isolate the Genomic DNA, the red coil. Later on you will see plasmids come into play with this lab as we inserted a section of the genomic DNA into the plasmid L4440.

//**Why did we isolate the DNA?**// We needed to ligate a section of the worms DNA that corresponds with the blister gene (Bli-1) into a plasmid. Therefore we needed the DNA from the worm. Through the lysing of the cell we are breaking open the cell, which contains unnecessary parts but also the genomic DNA. We then ran PCR of Genomic DNA on the cell in order to make copies to insert into the plasmid, L4440.

//**Preparation for Isolation**// Before this portion of the project began, it was important to have N2 worms actually growing on plates. The process to grow the worms is under the Worm Care section, edited by Dr. Radocchia. Once the worms were of the correct size we could begin the first step of the isolation process.

Worm Picks Lysis Buffer Sterile H₂0 10X Buffer 1.5 mL tubes PCR Tubes NGM-Lite Plates with OP-50 and Worms growing Freezer Heat Block Proteinase K **
 * //Tools for Isolation//

//**Worm Lysis**// Lysing the worm breaks open its "shell" and allows us to take the DNA. Although, in order to lyse the worms, we had to create the lysis buffer (ingredients below). Afterwards, we picked the worms and put them into tubes containing buffer and we froze them and heated them to break them open. The procedure is listed below.

//Ingredients for Lysis Buffer//
 * 5μL of proteinase K
 * 90μL of sterile H₂0
 * 10μL of 10X Lysis Buffer

// Procedure for Worm Lysis Buffer // ​(Diagrams displayed underneath for visual learners-Diagram 1)
 * 1) Add 90μL of sterile H₂0 with 10μL of 10X Buffer to create a 1X Lysis Buffer concentration
 * 2) Add 95μL of the 1X Lysis Buffer with 5μL of proteinase K

Diagram 1: How to create the Worm Lysis Buffer

// Procedure for Worm Lysis // (Diagrams displayed underneath for visual learners-Diagram 2)
 * 1) Put 10μL of the Lysis Buffer in each PCR tube used. (We created multiple tubes in order to have extra in case something went wrong).
 * 2) Using a copper pick that has been sterilized with alcohol, pick off 3-5 medium sized worms and place them into each PCR tube. (make sure each worm is alive-this can be checked by seeing if they writhe in the buffer).
 * 3) Centrifuge the worms if they are not all contained in the buffer.
 * 4) Freeze the tubes at -20°C for 10 minutes.
 * 5) Heat the tubes at 65°C for 60-90 minutes.
 * 6) Heat the tubes to 90°C for 20 minutes.
 * 7) Depending on whether or not you can immediately run the PCR, the genomic DNA contained in the tubes should be frozen until needed (to prevent denaturing).

Diagram 2: The procedure for isolating the DNA //**What really happened with the Lysis?**// To put it simply, the worms were placed in the tubes containing the buffer that broke open the cells with the assistance of the heating and freezing. While creating the buffer, it was important to achieve a 1X concentration, hence using 90μL of sterilized water. The next step was picking worms. Looking at the plate, Figure 1, one can see there are numerous sizes to the worms. It was important that the worms chosen were of medium to large size. This is so we knew the worm was at an adult stage and contained all of the DNA. The alive worms were picked off the plate with copper picks (Figure 2) so as not to gauge the agar but also because the worms are extremely small. Placing these worms into the lysis buffer thus began the lysing process. The freezing process and the immediate switch to the heat, along with the buffer, lysed the worms open and allowed us to remove its contents, the important one being the genomic DNA. Afterwards, the heating to 90°C denatured the enzyme proteinase K.

**//What's next?//** After the Lysis was completed, the tubes were ready for the PCR of Genomic DNA. The DNA of the //C. elegans// was not contained in the cell anymore and we were ready to make copies of the specific segment necessary to produce blisters. A thorough explanation is on Dr. Lee's page (the link above). In order to prove that the isolation was successful, we ran a gel of the PCR product (Figure 3). Because there was a band in lanes PCR 1, PCR 2, and PCR 3 it's proven that DNA was isolated successfully from //C. elegans.//

This ran successfully the first time through as seen in Figure 3, however, we ran into many problems later on. Luckily we made three tubes of lysed worms so we had enough to not have to restart from the beginning. When prepping for the entire lab we worked backwards figuring out amounts we needed for the last part to be successful. We decided since the procedure for isolating the DNA was so simple, that we created three tubes instead of two. When needed to redo a particular part of the experiment, because of either procedural errors or gels indicating that something was amiss, we were lucky enough to not have to start from the beginning. Figure 1: Worms seen at a magnification of 3X Figure 2: Copper Pick
 * // Conclusions //** //**/C**// **// omplications //**

Figure 3: PCR Gel to confirm PCR Product and Isolated Genomic DNA