Background on Genomic DNA Isolation and Purification
Generally, all methods involve the disruption and lysis of cells. This is followed sometimes by the removal of RNA (by RNAses, salt or other methods). Choosing which method to use will depend on many selection factors including: DNA is isolated from proteins by several methods including digestion of proteins by the enzyme proteinase K. Proteins are removed subsequently by salting-out, organic extraction, or binding of the DNA to a solid-phase support (such as an anion-exchange column or silica technology). DNA is finally recovered by ethanol precipitation or isopropanol precipitation. In general, the separation of DNA from cells and cellular components can be divided into four stages: 1. Cell disruption
2. Lysis of Cell
3. Removal of Proteins and Contaminants
4. Recovery of DNA
In some genomic DNA isolation protocols, stages 1 and 2 are combined. ISOLATION AND QUANTIFICATION OF GENOMIC DNA
Learning objectives for this lab:
¾ Understand the basic process behind a DNA isolation (extraction) procedure ¾ To get a “feel” for what DNA looks like
¾ To learn how to determine the concentration of DNA using spectrophotometry ¾ To learn how to make a standard curve
First: Read through the entire lab. Focus on the questions you will need to answer. Second: With your group, sketch out a flow chart of each step. Each person should have this in their notebook. Use diagrams/short sentences.
You will also learn how to determine the concentration of the DNA you isolate using a spectrophotometer. You will measure the absorbance of the DNA solution you isolate and compare it to a DNA solution of known concentration.
DNA Isolation Protocol
1. Weigh out 60 g of onion and mince it into smaller pieces using a razor blade (carefully!). You may use the scale at the back bench to check the weight of your pieces. 2. Put your minced onion and 50 ml of the cold citrate-saline buffer in the blender and blend on high for 30 seconds then low speed for 1 min – 2min. You want to ensure that there are no large chunks left.
3. Place 4 layers of cheesecloth over the top of a beaker. Pour the homogenized onion through the cheesecloth and into the beaker. You may have to pour a little, let it filter through and then pour more. You may also have to squeeze the homogenate through the beaker with your hand. 4. Add your filtered homogenate to a 50 ml centrifuge tube. If the homogenate is foamy, use a pipette to remove the foam and add the rest of the liquid homogenate. Use a wax pencil to mark the top level of the liquid in your tube and to identify it as belonging to your group.
5. Each group should now have a tube of filtered homogenate and your instructor will help ensure that all the tubes are balanced and load them into the refrigerated centrifuge. The tubes will be centrifuged at 6000 rpm for 10 min.
6. Remove your tube from the centrifuge being careful not to disturb the pellet. Gently pour off and discard the supernatant. Add fresh, cold citrate-saline half-way (ie. use half the amount this time) to the mark you made on the tube. Use a pipette to break up the pellet, you may also cover the top of the tube with a lid and shake or vortex until all of the pellet is resuspended. 7. Centrifuge again at 6000 rpm for 10 min. Spring 06
8. Again, discard the supernatant. This time, resuspend the pellet in 50 ml (use the mark on your tube) 2.6 M NaCl solution, using same method as in #6.
9. Centrifuge again at 6000 rpm for 10 min.
10. This time – pour the supernatant into a 250 ml beaker that has been sitting on ice. At this point, the supernatant contains the DNA in your solution. You will discard the pellet. KEEP THE DNA SOLUTION CHILLLED.
11. Add an equal volume (you can estimate) of ice cold ethanol to the lysate but you must do it by SLOWLY pouring the ethanol down the side of the beaker. The goal...
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