Examples of centrifugal separation of biological large molecules.

example (1) deproteinated RNA separation:
(i) sample homogeneity added 9 times the volume of ice-cold 20mM Tris-HCl (PH8.0) 1mM
EDTA
.
10% of the 1/10 volume is added to the
. SDS
, then add iso-volume (phenol: chloroform: isopropyl alcohol) (volume ratio 50: 50: 2) solution and contain 0.1% of the 8-hydroxyquine solution fully mixed to emulsify it.
(iii) above the solution at 10,000xg centrifugation for 10 minutes.
(iv) out of the upper liquid and repeat the (ii) (iii) process once.
( v ) Add 1/10 volume of ammonium acetate to the second centrifugal liquid, mix it well, add the ethanol with double volume, and set aside for more than two hours at -20 degrees C.
RNA precipitation in 10 minutes with a centrifugation of 10,000xg 5 x C.
(vii) evaporates
remaining ethanol
the sediment with n2 gas that is dried and dried.
(viii) dissolves RNA in 20mMTris-HCl (PH8.0), 1mMEDTA, at -20 degrees C for low temperature storage.
example (2) isolated the prosury from E. coli
. DNA
:
(i) Solution Preparation:
Solution I: 50mM Glucose, 10mM (EDTA), 25mM Tris-HCl (PH8.0)
Solution II: 0.2M NaOH, 1% SDS
Solution III: 3M acetate (PH4.8)
TE buffer: 10mMTris-HCl (PH7.4), 2mM EDTA
(ii) E. Coli
culture
1 liter (large proportional configuration)
(iii) 1 liter culture fluid at 4,000 rpm (approximately 2,000xg), 5 degrees C centrifugation for 20 minutes, to obtain bacterial precipitation.
( iv ) with 100 ml of icy solution I "wash" precipitation, centrifugation again, 4,000rpm, 5 degrees C, 20 minutes.
(v) adjust the second centrifugal precipitation to a uniform suspension with 20 ml of ice-cooled solution I.
2mg lysolysase is added to each ml suspension and placed in an ice bath at 0 degrees C for 10 minutes.
40ml solution II to the ice bath (vii), stir gently and leave in the ice bath for 5 minutes.
30ml Solution III to the ice bath for 20 minutes to allow the
protein
most of the subside.
(ix) centrifuges
15 minutes at 10,000 rpm (about 10,000xg) on a high-speed frozen
centrifuge
, 5 degrees C.
(x) carefully pour over the top (without disturbing the precipitation) and add 55 ml of isopropyl alcohol to the precipitation, i.e. to obtain a crude granule DNA solution.
(xi) crude DNA fluid is set aside at -20 degrees C for more than 15 minutes, then centrifuged at 12,000 rpm (approximately 15,000xg) for 5 minutes and poured over for dehydration.
isopropyl alcohol isopropanol in a vacuum, 18 ml of disinfected TE buffer is added to the precipitation, 0.65 ml is added after the precipitation dissolves, and 1% E.B.
. ( xiii ) the above solution per ml to add analysis pure CsCl 1 g, so that it is completely dissolved, the detection solution refractive index should be 1.3890 (density ≈ 1.587)
(xiv) with the above solution in accordance with the reference (6) as described in the method of mass DNA separation.
(xv) centrifugation can show DNA bands (linear DNA bands and proton DNA bands, RNA precipitation at the bottom, protein floating on the liquid surface) in 300nm UV light.
(xvi) is used in the literature (1) to remove the prosaic DNA, remove the E.B. dialysis method or desalinate column to remove CsCl.
example (3) the relationship between the separation of DNA with a CsCl gradient and the determination of its composition (% G-C)
(i) CsCl solution density D and the weight percentage concentration P of CsCl in the solution: D=138.11/ ( 137.1 48-P)
(ii) prepares gradient fluids with an initial density of 1.706 g / ml, which should be configured as follows
4.85 g CsCl (analytical pure)
50 μ l, 1.0M Tris-HCl (PH7.4)
20 μ l, 0.2M EDTA (PH7.4)
0.5ml DNA sample
3.3ml refraction water
(iii) light refractometer to detect CsCl The initial density D, with a measured light refractive index of RI, should be 1.4000 if the detection value is greater than this value, with a D-10.8601 × RI-13.4974
(iv) measured RI. The re-aggravated steamed water capacity is V (ml), the total capacity of the gradient fluid is G (ml), the total capacity of the gradient fluid is 1.52 × G × (D as measured -D required), and if the detection value is less than 1.4000, then the reinforcement state CsCl (W) g
W is 1. 32 × G × (D requires -D assay)
(v) Retest the refractive index of the solution until the RI is adjusted to 1.4000
(vi) fills the configured liquid with a one-time fast-sealing centrifuge tube of 5 ml and seals it with a sealer.
( vii ) fixed angle turn 150,000xg , 20 degrees C centrifugation 50 to 55 hours (approximately 35,000 rpm) or vertical tube turn 300,00 0xg, 20 degrees C centrifugation 12 hours (approximately 55,000rpm)
(viii) centrifugation using a gradient meter or manual collection into 50 tubes (100 μ l each).
(ix) measures the refractive index to 4 bits per tube sample, the sample temperature should be kept at 20 degrees C during testing, and the density D of each tube sample should be calculated based on the refractive index RI.
(x) Add 1 ml of reheated water to each tube and measure the light density (OD) of each tube at 260nm wavelength, and if the DNA has been labeled
isotope
then the collected samples are separately measured by a liquid flashing meter to determine the radiation value.
(xi) is curved in ordinates (from the bottom of the tube to the surface) of the centrifuge tube capacity, with the OD value or isotope radiation count value measured by each tube as the ordinate, respectively. DNA positioning can be done by finding the peak position of the curve.
(xii) uses the following calculation of DNA composition:
% (G-C) - (D-1.66) / 0.098 × 100
the calculation is more accurate if standard DNA can be added to centrifugal samples.
.