Purification of proton DNA

(i) Polyethyl glycol precipitation method prosurage DNA
The method described here (R.Tresman, personal communication) has been effectively used to purify the granulated DNA prepared by the legal system of alkali lysing.
1) Transfer the
nucleic acid
solution (step 9 above) into a 15 ml Corex tube, add 3 ml with an ice-cooled 5mol/L LiCl solution, mix well, and turn the head of the Sorvall SS34 (or its equivalent) at 4 degrees C at 10 000 rpm/separation for 10 minutes. LiCl precipitates polymer RNA.
2) Transfer the upper clear to another 30 ml Corex tube, add the same amount of isopropyl alcohol, mix well, with SorvallSS34 turn head (or its equivalent tip) at room temperature at 10 000 rpm / separation heart 10 cents, recovery of the precipitated nucleic acid.
3) Carefully remove the upper clear, open the tube opening, and turn the tube upside down so that the last remaining droplets are exhausted. Wash the sediment and pipe wall with 70% ethanol at room temperature, drain the ethanol, suck off all the droplets attached to the pipe wall with the Bajad straw connected to the vacuum device, open the tube opening and place the tube nephew on the paper towel for a few minutes, so that the last remaining trace ethanol evaporates.
4) Dissolve the precipitation with TE (pH8.0) of 500 μl of pancreatic RNA enzyme (20?g/ml) containing DNA-free enzymes, transfer the solution to a trace centrifuge tube and place it at room temperature for 30 minutes.
5) plus 500 sl containing 13% (w/v) polyethyl glycol (PEG 8000) of 1.6mol/L NaCl, fully mixed, with trace
centrifuges
centrifuges at 4 degrees C at 12000g centrifuges for 5 minutes to recover the granule DNA.
6) sucks out the upper sequestration and dissolves the granulate DNA precipitation with 400 μl TE (pH8.0). With phenols, phenols: chloroform, chloroform each pumped 1 time.
7) Transfer the water phase to another trace centrifuge tube, add 100 μl 10mol/L ammonium ethanol, mix well, double the volume (about 1 ml) of ethanol, place at room temperature for 10 minutes, centrifuge at 4 degrees C at 12,000g for 5 minutes, in order to recover the precipitated granulated DNA.
8) suction, plus 200 sl at 4 degrees C to 12,000g centrifugal 2 minutes.
9) sucks up the upper clear, opens the tube, and places the tube on the experimental table until the last visible trace of ethanol evaporates.
10) Dissolve the precipitation with 500 slTE (pH8.0) 1:100 dilution (OD260 after using TE (pH8.0)) to calculate the concentration of the prosurage DNA (1OD260 x 50 sg prosulte DNA/ml), and then store the DNA at -20 degrees C.(ii) chlorpyrifoethylene-brominated ethyl bromide ingot gradient balance centrifugation method purifying closed-loop DNA 1. Continuous gradient
1) measures the volume of the DNA solution, precisely adding solid CsCl in lg/ml, and heating the solution to 30 degrees C to help dissolve. Gently mix the solution until the salt dissolves.
2) Add 0.8 ml of ethyl bromide ingot solution (10 mg/ml soluble in water) to every 10 ml of DNA solution; The final density of the solution should be 1.55g/ml (the refractive index of the solution is 1.3860) and the concentration of ethyl bromide ingots should be approximately 740 mg/ml. Ethyl bromide ingot storage fluid should be stored in a light-absorbing container (e.g. bottles completely wrapped in tin foil). Store at room temperature.
3) At room temperature with Sorvall SS34 head (or its equivalent turn head) at 8000 rpm / separation of the heart for 5 minutes, the scale-like slag floating on top of the solution is a complex formed by ethyl bromide ingots and bacterial proteins.
4) Transfer the bright red solution under the pontoon to a centrifugal tube (Beckman Quick-seal or equivalent centrifuge tube
) for Beckman Ti65 or Ti50, Ti65, or Ti70 corner turns (or the equivalent of them) with a Pasteur straw or disposable syringe with a large needle. Fill the rest of the tube with light paraffin oil and seal.
5) the resulting density gradient at 20 degrees C is 45,000 l/separation heart 16 hours (VTi65 turn), 48 hours at 45,000 turn/separation heart (Ti50 turn), 24 hours at 60,000 s/separation heart (Ti65 turn) or 60,000 s/separation heart 24 hours (Ti70.1 turn). In normal light, two DNA zones can be seen at the center of the ladder, the upper band material is usually less, consisting of line-like bacterial (chromosome) DNA and ring-like granule DNA with inlets: the lower zone belt is composed of closed-loop granule DNA. The crimson precipitation at the bottom of the tube is a brominated ethyl ingot RNA complex located between the CsCl solution and paraffin oil,
protein
. The CSCl-Brominated Ethyl Bromide ingot gradient in Beckman Quick-Seal centrifugal tubes can accommodate 4 mg of closed-loop granulated DNA without overloading. If a longer number of prosthytes are present, they will expand to a wide bandwidth and overlap with chromosomal DNA. This problem only occurs when the granules are replicated to extremely high levels, as long as the particle extract is divided into two gradients. In the event of a load, the entire DNA region can be collected at a high level, as long as the production of the granule extract into 2 gradients can be resolved. In the event of overloading, the entire DNA zone band can be collected, and the volume is adjusted to 15 ml with a CSCl solution (ρ x 1.58g/ml) to re-centrifuge in two centrifugal tubes to balance the DNA.
6) Collect DNA bands. To minimize the chance of contamination by inserting the 21st subsurfic injection needle into the top of the tube, the upper band (chromosome DNA) is collected using the 18th subsurfic injection needle as follows: carefully wipe the outer wall of the tube with ethanol to remove any grease, and then attach a piece of Soctch tape to the outer wall of the tube. Insert the 18th leather pin (with its slope up) into the tube through Soctch tape so that the bevel opening of the needle is located just below and parallel to the chromosomal DNA zone. Collect the viscous DNA into a disposable tube, inject the non-end of the needle under the skin with a styling clay block, and leave the second needle in place. Insert the third subsurfic injection needle (No. 18) through Soctch tape and collect the lower granule DNA band into a glass or plastic tube.
2. Non-continuous gradient
This method is to layer solutions containing different concentrations of CSCl into centrifugal tubes, which can accelerate the formation of CSCl gradients and reduce centrifugal time to 6 hours.
1) Add 125gCsCl to 167ml (pH8.0) to make a CsCl solution (ρ x 1.47g/ml).
2) Add 8 ml of cesium chloride solution to the Beckman Quick-Seal centrifuge tube (or tube equivalent to it) and set aside to wait for step 8 to be used.
3) If necessary, the volume of the proton DNA solution can be precisely adjusted to 3 ml using TE (pH8.0) as appropriate.
4) Add 8.4 gCsCl to the proton DNA solution, warm the solution to 30 degrees C to promote salt dissolution, carefully mix the solution until the salt dissolves.
5) weigh the weight of the solution and add TE (pH8.0) until the weight of the solution is exactly 13.2g, with
balance
weighing should pay attention to remove the weight of the tube.
6) Add 0.8 ml of ethyl bromide ingot solution (10 mg/ml soluble in water) and quickly mix the solution until the dye is evenly dispersed, at which point the volume of the solution valve should be approximately 7.5 ml. Ethyl bromide ingot storage fluid should be stored at room temperature in a light-absorbing container (e.g. bottles completely wrapped in tin foil). Caution: Ethyl bromide is a strong mutagent and is moderately toxic.
should wear gloves and gloves when touching solutions containing
dye
.
7) At room temperature with Sorvall SS34 head (or equivalent) at 8000 rpm / separation of the heart for 5 minutes, the scale-like slag floating on the liquid surface is a complex of bromide ethyl ingots and bacterial proteins.
8) Place the -22.86cm Bardes straw in a centrifugal tube equipped with step 2) CsCl, and the suction head should touch the bottom of the tube. Carefully add the bright red solution prepared by step 7 (from under the slag) to the tube with a straw so that the sample layer CSCl solution (1.47g/ml) is below. If necessary, fill the centrifugal tube and seal the CSCl solution (ρ x 1.47g/ml) prepared in Step 1 as appropriate.
9) Place the sealed tube (along with the corresponding balancing tube) in Beckman Ti70.1 or Sorvall 65.13 turnheads (or equivalent turns) and centrifuge the density gradient for 6 hours at 20 degrees C at 60,000 rpm.10) Recovery of closed-loop proton DNA zones
(iii) removal of ethyl bromide ingots from purified granulated DNA
the method below is equally effective for the removal of ethyl bromide ingots from dna that has been balanced with centrifugal purification of cesium chloride-bromide ingots.
method:
organic
solvent extraction
Be careful: ethyl bromide ingot is a strong mutagent agent, and has moderate toxicity. Wear gloves when touching solutions containing the dye. These solutions are then purified using the method described below.
1) Put the DNA solution into a glass or plastic tube and add the same volume of water to saturate 1-butanol or isosterol.
2) Oscillation mixes two phases.
3) 3 minutes at room temperature with a desktop centrifuge at 1500 ropm/separation.
4) Move the lower water phase into a clean glass or plastic tube using a Bardsard straw.
5) Repeatedly pumping (step 1)-4) 4-6 times until the pink disappears from both the water phase and the organic phase.
6) Remove CsCl from dna solution by any of the following methods: rotating dialysis via microcontrol (Amicon), te (pH8.0) dialysis for 24-48 hours, and liquid change several times or dilute with 3 times the volume of water and 4 The sedimentation of D15 minutes with 2x volume ethanol (i.e. the final volume is equivalent to 6 times the original undiluted volume) and then centrifugation of 15 centrifuges at 4 degrees C dissolves the precipitated DNA in approximately 1 ml TE (pH8.0). CsCl precipitates if the ethanol solution of the DNA is placed at -20 degrees C.
7) to determine the OD260 value of the final solution of DNA, calculate the concentration of DNA, the DNA into small parts stored at -20 degrees C. If the final DNA contains a significant amount of ethyl bromide (color judgement), phenols, phenols: chloroform are drawn 1 time each, and then the DNA is precipitated with ethanol.
(iv) Purification treatment of ethyl bromide solution
Be careful: Ethyl bromide ingot is a strong mutagent agent, and has moderate toxicity, take the solution containing this dye must wear gloves, these solutions should be used in accordance with the method described below for purification treatment.
1. Purification treatment of ethyl bromide ingot thick solution (i.e. concentration >0.5gm/ml of ethyl bromide sink solution)
method: With the determination of less mentabacteria-particles, this method (Lunn and Sanaone, 1987) can reduce the mutagel activity of ethyl bromide ingots to about 1/200.
1) Add enough water to reduce the concentration of ethyl bromide ingots to less than 0.5 mg/ml.
2) Add 5% phosphoric acid in 0.2 volume and 0.12 sodium nitrite in each new preparation, mixing well. : The pH of the solution should be less than 3.0. Commercially available phosphoric acid is generally 50% solution, corrosive, should be carefully operated, must be diluted now. Sodium nitrite solution (0.5mol/L) is available for water dissolving 34.5g of sodium nitrite and until the final volume of 500ml.
3) After 24 hours of room temperature, add a significant excess of 1 ml/L sodium bicarbonate. To some, the solution can be discarded. Return : The pH of the solution should be less than 3.0. Commercially available phosphoric acid is generally 50% solution, corrosive, should be carefully operated, must be diluted now. Sodium nitrite solution (0.5mol/L) is available for water dissolving 34.5g of sodium nitrite and until the final volume of 500ml.
3) After 24 hours of room temperature, add a significant excess of 1 ml/L sodium bicarbonate. To some, the solution can be discarded. (v) The removal of RNA
from proton DNA products for certain purposes (e.g., the use of BAL31 into the living or the use of T4 phage poly
nucleotides
-excited markers at the 5' end of the restricted cut fragment of the granule DNA) must obtain RNA-free DNA products. Although the weight of such RNA contaminants is small in the granulated DNA prepared by the chlorinated cesium-ethyl chloride ingot gradient balance centrifugation, the number of RNA molecules in it may be considerable and can account for the proportion of sand tolerance neglect in all 5' ends that limit the enzyme's digestion reaction. RNA can be removed from the granules by the following methods. 2. The proton DNA is prepared by Bio-Gel A-150m or Sepharose CL-4B
1).
2) has an equal volume of TE (pH8.0) after balancing the phenol pumping 1 time.
3) spread up to 1 ml of water in a balanced B balanced by TE (pH8.0) and 0.1% SDS