The method used for the isolation of large scale cosmid and plasmid DNA is an. The ethidium bromide stained.
DNA bands, equilibrated within the cesium chloride density. UV light and. the lower band is removed with a 5 cc syringe. Pick a colony of bacteria harboring the plasmid or cosmid DNA of interest. X 7. 5 mm Falcon tube containing 2 ml of LB media supplemented with the. C 8- 1. 0 hours with shaking at 2. Harvest the cells by centrifugation at 7. RC5- B using the GS3 rotor. Over 5 hours of Astronomical Image Processing Tutorilas are available on DVD. The Camtasia Studio video content presented here requires JavaScript to be enabled and the latest version of the Macromedia Flash Player. Questions of biochemistry with answers of all chapter 1. What are the chemical elements that form most of living biological matter? The chemical elements that form most of the molecules of living. Kyrie Irving had a monster Game 5 by dropping 41 points at Oracle Arena to keep the Cavs alive. He wore the Nike Kyrie 2 'Gradient'. Resuspend the cell pellets in a total of 7. GET/Lysozyme solution (3. Add a total of 1. Add 1. 05 ml of 3. M Na. OAc, p. H 4. Clear the lysate of precipitated SDS, proteins, membranes, and chromosomal. DNA by pouring through a double- layer of cheesecloth. Pool the cleared supernatants into to a clean beaker, add one- fourth volume. PEG/0. 5 M Na. Cl, swirl to mix, and incubate in an ice- water bath for 1- 2. Collect the PEG- precipitated DNA by centrifugation in 2. C in the RC5- B using the GSA. Dissolve the pellets in a combined total of 3. TE buffer, 5 ml. of 5 mg/ml ethidium bromide, and 3. Var Lac Oid Chemical. Co., Inc.) (final concentration of cesium chloride should be 1 g/ml). Transfer the sample into 3. Centrifuge at 6. 0,0. C in. the Sorvall OTD- 7. B ultracentrifuge (Du. Pont) using the T- 8. Visualize the ethidium bromide stained DNA under long- wave UV light, and. DNA band using a 5 cc syringe and a 2. To remove the ethidium bromide, load the DNA sample onto an equilibrated. Dowex column, and collect 0. Pool fractions with an A2. Corex glass. tubes, add one volume of dd. H2. O, and ethanol precipitate by adding 2. Resuspend the DNA in 1. TE buffer. For diatomaceous earth- based purification. Pool the supernatants from step 6 into 5. DNase- free. RNase A and RNase T1 such that the final concentration of RNase A is 4. RNase T1 is 4. 0 U/ml. Add an equal volume of isopropanol and precipitate at room temperature for. Resuspend each DNA pellet in 2. TE buffer, and add 4. HCl (1. 00 mg/ml) to each bottle. Decant the supernatant, resuspend each pellet in 4. Decant the supernatant, resuspend each pellet in 4. Decant the supernatant and dry the pellet in a vacuum oven. Resuspend the pellet in 2. TE buffer, and elute the bound DNA. C for 1. 0 minutes with intermittent. Remove the diatomaceous earth by centrifugation at 9,0. RC5- B using the GS3 rotor. Combine the DNA- containing supernatants and precipitate the DNA in 3. Corex glass tubes adding 2. Resuspend the dried DNA pellet in 2 ml of 1. TE buffer and assay for. A midi- prep double- stranded DNA isolation has been developed to generate a. DNA for several Sequenase. Pick a colony of bacteria harboring the plasmid DNA of interest into a 1. X. 7. 5 mm Falcon tube containing 3 ml of 2x. TY media supplemented with the. C 8- 1. 0 hours with shaking at 2. Harvest the cells by centrifugation at 3. Beckman GPR tabletop centrifuge and decant the. Resuspend the cell pellets in 2 ml of GET/Lysozyme solution, add 4 ml of. Add 4 ml of 3. M Na. OAc, p. H 4. 8, gently mix by swirling, and incubate in an. Clear the lysate of precipitated SDS, proteins, membranes, and chromosomal. DNA by pouring through a double- layer of cheesecloth into a new 5. Decant the supernatant to a 5. DNase- free RNase A and incubate in a 3. C. water bath for 3. Add 7 ml (equal volume) of de- fined diatomaceous earth in guanidine- HCl (2. DNA to bind at room temperature for 5 minutes with. Decant the supernatant, resuspend in 7 ml of diatomaceous earth- wash buffer. Decant the supernatant, resuspend in 7 ml of acetone, and centrifuge as. Decant the supernatant and dry in a vacuum oven. Resuspend the pellet in 0. TE buffer, and elute the bound DNA. C for 1. 0 minutes with intermittent. Remove the diatomaceous earth by centrifugation at 3,0. Beckman GPR tabletop centrifuge. Transfer the supernatant to a 1. Resuspend the dried DNA pellet in 4. TE buffer and assay for. The standard method for the miniprep isolation of plasmid DNA includes the. Pick a colony of bacteria harboring the plasmid DNA of interest into a 1. X. 1. 00 mm Falcon tube containing 6 ml of TB media supplemented with the. C 1. 6- 1. 8 hours with shaking at 2. Harvest the cells by centrifugation at 3. Beckman. GPR tabletop centrifuge and decant the supernatant. Resuspend the cell pellets in 0. TE- RNase solution (5. TE buffer. containing 4. RNase A; some also add RNase T1 to a final concentration of. U/ul) by gentle vortexing, add 0. Add 0. 2 ml of 3. M Na. OAc, p. H 4. Clear the lysate of precipitated SDS, proteins, membranes, and chromosomal. DNA by centrifugation at 1. C. Transfer the supernatant to a fresh 1. For standard alkaline lysis purification. Precipitate the DNA by adding 1 ml of 9. DNA pellet in 1. 00- 2. TE buffer. Add 1 ml of de- fined diatomaceous earth in guanidine- HCl (2. DNA to bind at room temperature for 5 minutes with occasional mixing. Turn on the vacuum pump and adjust the vacuum level to 8 in. Hg, let the. membrane dry for 1 minute, and then release the vacuum. Pour the well mixed samples into the wells of the Prep- A- Gene manifold and. Hg until all the liquid is filtered through. Wash the samples four times with 2. Reduce the vacuum to 5 in. Hg before turning the vacuum off at the. Hg, and let the membrane dry for 1 minute. Turn off the vacuum at the stopcock and remove the collection rack. Prepare an early log phase culture of JM1. Ehrlenmeyer. flask containing 5. TY with a glycerol stock of JM1. C water bath, with no. Harvest the cells by centrifugation at 7. RC5- B using the GS3 rotor. Resuspend the cell pellets in a total of 1. X STB buffer by gently teasing the pellet with a spatula. Add 4. 8 ml of 5. TTE buffer (1. 2 ml for each bottle) and 2 ml of RNase A. Clear the lysate of precipitated SDS, proteins, membranes, and chromosomal. DNA by pouring through a double- layer of cheesecloth. Add 6 ml of 5 mg/ml ethidium bromide, and cesium chloride such that the. Transfer the sample into 3. TE buffer and cesium chloride, remove air bubbles. Centrifuge at 6. 0,0. C in. the Sorvall OTD- 7. B ultracentrifuge using the T- 8. Visualize the ethidium bromide stained DNA under long- wave UV light, and. DNA band using a 5 cc syringe and a 2. To remove the ethidium bromide, load the DNA sample onto an 1. Dowex AG. (Bio. Rad) column, equilibrated as before, and collect 0. Pool fractions with an A2. Corex glass. tubes, add one volume of dd. H2. O, and ethanol precipitate by adding 2. Resuspend the DNA in 1. TE buffer. This isolation procedure (2. M1. 3- based templates to be used in Sequenase. Prepare an early log phase culture of JM1. M1. 3- based. plaques with sterile toothpicks into 1. X 7. 5 mm Falcon tubes containing 1. Transfer the culture to 1. C. Pipette the top 1 ml of supernatant to a fresh 1. PEG/2. 5 M Na. Cl to precipitate the phage particles. Centrifuge for 1. C to. collect the precipitated phage. Resuspend the pellet in 1. M Tris- HCl, p. H 7. TE- saturated phenol. Extract the DNA with phenol and twice with ether, as discussed above, and. Resuspend the dried DNA in 6 ul of 1. TE for use in single- stranded. Sequenase. Prepare an early log phase JM1. TY, as above. Using sterile toothpicks, transfer individual M1. X 7. 5 mm. Falcon tubes containing 1 ml early log phase cell cultures, and incubate for. C with shaking at 2. Separate the bacterial cells from the viral- containing supernatant by. C. The Biomek will distribute two 2. Dynatech). Cover the plate with an acetate plate sealer and incubate at room. Pellet the precipitated phage by centrifuging the plate at 2. Beckman GPR tabletop centrifuge. Return the plate to the tablet, and the Biomek will robotically add 2. PEG: TE rinse solution to each well. Return the plate to the tablet, and the Biomek will add 7. TTE. solution to each well. The Biomek then will robotically pool the contents from each pair of wells. Incubate the tubes at 8. C for 1. 0 minutes to denature. Ethanol precipitate the DNA by adding 5. Resuspend the DNA templates in 2. TE buffer. Transfer 1. Prior to adding the fluorscent terminator. C. Pick colonies using a toothpick into 1. TB with TB salts containing appropriate. Harvest cells by centrifugation at 1. Turn on Biomek, begin the program DSISOL2 and set up the Biomek as indicated in. Specifically, you should put TE- RNase solution. M KOAc. p. H 4. 8 in the third module. Place the 9. 6 well block containing cells onto the Biomek tablet at the position. Press ENTER to continue with the program. First the Biomek will add 1. TE- RNase solution to the cell pellets and mix to. Next, the biomek will add 1. The biomek then will mix the cell suspension again, transfer the entire volume. The biomek will add 1. M KOAc, p. H 4. 8 to the wells of the filter plate and. The supernatant collected in the 9. DNA and must be ethanol. C. for at least 3. Centrifuge for 2. Beckman GPR centrifuge. Decant and wash with 5. Decant the supernatant, drain inverted on a paper towel. Resuspend in 5. 0 ml 1. TE for use in dye primer or dye terminator sequencing. Genomic DNA isolation is performed according to the FBI protocol (2. Blood samples typically were obtained as 1 ml of whole blood stored in EDTA vacutainer tubes frozen at - 7. C. Thaw the frozen samples, and to each 1 ml sample, add 0. X SSC buffer, and mix. Remove 1 ml of the supernatant and discard into disinfectant. Add 1 ml of 1. X SSC buffer, vortex, centrifuge as above for 1 minute, and. Add 3. 75 ul of 0. M Na. OAc to each pellet and vortex briefly. Then add 2. 5 ul. SDS and 5 ul of proteinase K (2. H2. O) (Sigma P- 0. C. Add 1. 20 ul phenol/chloroform/isoamyl alcohol and vortex for 3. Carefully remove the aqueous layer to a new 1. C. Centrifuge for 2 minutes at 1. Add 1. 80 ul 1. 0: 1 TE buffer, vortex, and incubate at 5. C. for 1. 0 minutes. Add 2. 0 ul 2 M sodium acetate and mix. Decant the supernatant and rinse the pellet with 1 ml of 8. Decant the supernatant, and dry the pellet in a Speedy- Vac for 1. Resuspend the pellet by adding 2. TE buffer. Roe, Department of Chemistry and Biochemistry. The University of Oklahoma, Norman, Oklahoma 7. Gradient. XTerminator. The Camtasia Studio video content presented here requires Java. Script to be enabled and the latest version of the Macromedia Flash Player. If you are you using a browser with Java. Script disabled please enable it now. 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