1. Perform the necessary calculations for the restriction digestions that you wi
ID: 201890 • Letter: 1
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1. Perform the necessary calculations for the restriction digestions that you will set up this week How many digest reactions wilyou set up? 2. What is the function of the enzyme RNase? Why is it important that RNase is included when purifying our plasmids? You will perform this procedure for each of the liquid cultures that you grew overnight 1. Transfer 1.4 ml of cells from your overnight culture into a mcfg tube. 2. Spin at max speed for 3 minutes. Discard the supernatant in the cell waste flask 3. Resuspend the pellet in 250 1 of Solution l/RNase A. Mix well by pipetting up and down. 4, Add 250 1 of Solution II and gently invert the tube 4-6 times to mix. Do not vortex, as this will shear the genomic DNA into small pieces that will then be isolated with the plasmid DNA. If necessary, continue inverting the tube until the solution becomes viscous and slightly clear. Do not allow this lysis reaction to proceed for more than 5 min. Solution Il contains NaOH to lyse cells. CLOSE the bottle of Solution Il immediately after use so it does not become acidified by contact with CO2 in the air S. Add 350 ul of Solution Ill and invert the tube immediately but gently 4-6 times 6. Spin at max speed for 10 minutes. This spin will pellet the cell wall, genomic DNA (attached to the cell wall), and other cell junk. The plasmid DNA will remain in the supernatant. 7. Pipet the supernatant onto the column and centrifuge for 1 minute at max speed. Discard the flow through. The DNA is now adsorbed to the silica membrane in the column. 8. Add 500 ul of HBC Buffer and centrifuge for 1 minute. Discard the flow through. 9. Add 700 of DNA Wash Buffer and centrifuge for 1 minute. Discard the flow through and 8. Add 500 1 of HBC Buffer and centrifuge for 1 minute. Discard the flow through. 9. Add 700 1 of DNA Wash Buffer and centrifuge for 1 minute. Discard the flow through and centrifuge for another 1 minute to remove any residual ethanol from the column. 10. Place the column in a clean mcfg tube. Elute DNA by adding 50 1 of Elution Buffer to the center of the column. Let stand for 1 min, then centrifuge for 1 min. Mcfg tops MUST BE REMOVED-but save the tops for when you store your DNA Il. Restriction digestions (Set up 3: B, W1, and W2) A. Information for calculations Each reaction should be 20 l total Each reaction needs to include buffer E (supplied as a 10x stock) Use 5 1 of your purified plasmid DNA Each reaction needs 5 units of EcoRI (stock solution is 5 units/ Balance the volume of each reaction with sterile water B. Set up the digest reactions according to your calculations. Incubate at 37"C overnight. Be sure that your tubes are labeled clearly so that you can find them in the freezer next weeklExplanation / Answer
1.Digestion reaction
Total: 3 (tubes can be labeled as B,w1,w2)
For each one calculation
Buffer E 2ul
Pure plasmid 5ul
EcoRI 1ul
Sterile water 12ul
Total volume 20ul
2. RNase A is an important enzyme for the removal of RNA for RNA free DNA purification reactions such as plasmid DNA purification and genomic DNA purification, RNA removal from recombinant protein preparations, Ribonuclease protection assays, mapping single-base mutations in DNA/RNA.
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