388:
protocols advise storing DNA at low temperature at this point, but there are also observations that it may not improve DNA recovery, and may even lower precipitation efficiency while using over-night incubation time. Therefore, good efficiency can be achieved at room temperature, but when possible degradation is taken into account, it is probably better to incubate DNA on wet ice. Optimal incubation time depends on the length and concentration of DNA. Smaller fragments and lower concentrations will require longer times to achieve acceptable recovery. For very small lengths and low concentrations over-night incubation is recommended. In such cases use of carriers like
380:
676:
411:). Time and speed of centrifugation has the biggest effect on DNA recovery rates. Again smaller fragments and higher dilutions require longer and faster centrifugation. Centrifugation can be done either at room temperature or in 4 °C or 0 °C. During centrifugation precipitated DNA has to move through ethanol solution to the bottom of the tube, lower temperatures increase
61:
441:
can also be used instead of ethanol; the precipitation efficiency of the isopropanol is higher making one volume enough for precipitation. However, isopropanol is less volatile than ethanol and needs more time to air-dry in the final step. The pellet might also adhere less tightly to the tube when
423:
In the next step, 70% ethanol is added to the pellet, and it is gently mixed to break the pellet loose and wash it. This removes some of the salts present in the leftover supernatant and bound to DNA pellet making the final DNA cleaner. This suspension is centrifuged again to once again pellet DNA
362:
is much less polar than water, with a dielectric constant of 24.3 (at 25 °C). This means that adding ethanol to solution disrupts the screening of charges by water. If enough ethanol is added, the electrical attraction between phosphate groups and any positive ions present in solution becomes
387:
DNA is precipitated by first ensuring that the correct concentration of positive ions is present in solution (too much will result in a lot of salt co-precipitating with DNA, too little will result in incomplete DNA recovery) and then adding two to three volumes of at least 95% ethanol. Many
351:
The same mechanism operates in the case of negatively charged phosphate groups on a DNA backbone: even though positive ions are present in solution, the relatively weak net electrostatic force prevents them from forming stable ionic bonds with phosphates and
332:
415:
of the solution and larger volumes make the distance longer, so both those factors lower efficiency of this process requiring longer centrifugation for the same effect. After centrifugation the supernatant solution is removed, leaving a
367:
and DNA precipitation. This usually happens when ethanol composes over 64% of the solution. As the mechanism suggests, the solution has to contain positive ions for precipitation to occur; usually Na, NH
632:
556:
232:
205:
243:
158:
131:
178:
420:
of crude DNA. Whether the pellet is visible depends on the amount of DNA and on its purity (dirtier pellets are easier to see) or the use of co-precipitants.
476:
471:
605:
520:
647:
571:
456:
340:
around it. This fact makes water a very good solvent for charged compounds like salts. Electric force which normally holds salt
598:
696:
503:
666:
403:
During incubation DNA and some salts will precipitate from solution, in the next step this precipitate is collected by
432:
701:
353:
348:
is weakened in the presence of water allowing ions to separate from the crystal and spread through solution.
336:
At an atomic level, the reduction in the force acting on a charge results from water molecules forming a
404:
210:
183:
88:
327:{\displaystyle F={\frac {1}{4\pi \varepsilon _{r}\varepsilon _{0}}}{\frac {q_{1}q_{2}}{r^{2}}}}
506:, Melbourne, Australia; David Russell, University of Texas Southwestern Medical Center, Dallas
235:
73:
40:
207:(also called relative static permittivity) of the medium in the denominator of the equation (
408:
103:
136:
109:
499:
428:
337:
92:
80:
backbone. Its polarity makes it water-soluble (water is polar) according to the principle
65:
680:
451:
397:
163:
28:
427:
Finally, the pellet is air-dried and the DNA is resuspended in water or other desired
690:
379:
466:
438:
364:
345:
675:
461:
81:
36:
633:"Ethanol Precipitation: Ammonium Acetate as an Alternative to Sodium Acetate"
557:"Ethanol Precipitation: Ammonium Acetate as an Alternative to Sodium Acetate"
412:
77:
599:
bitesizebio.com The Basics: How
Ethanol Precipitation of DNA and RNA Works
393:
359:
341:
44:
424:
and the supernatant solution is removed. This step is repeated once.
96:
32:
60:
59:
106:, which can be used to calculate the force acting on two charges
389:
431:. It is important not to over-dry the pellet as it may lead to
87:
Because of the high polarity of water, illustrated by its high
95:
are considerably lower in aqueous solution than they are in a
24:
20:
498:
Molecular
Cloning: A Laboratory Manual (Third Edition) by
91:
of 80.1 (at 20 °C), electrostatic forces between
664:
246:
213:
186:
166:
139:
112:
326:
226:
199:
172:
152:
125:
407:in a microcentrifuge tube at high speeds (~12,000
19:is a method used to purify and/or concentrate
8:
477:Spin column-based nucleic acid purification
316:
305:
295:
288:
279:
269:
253:
245:
218:
212:
191:
185:
165:
144:
138:
117:
111:
435:of DNA and make it harder to resuspend.
378:
671:
488:
7:
550:
548:
514:
512:
494:
492:
68:of a sodium ion dissolved in water
14:
180:by using the dielectric constant
674:
504:Peter MacCallum Cancer Institute
383:A laboratory tabletop centrifuge
227:{\displaystyle \varepsilon _{0}}
200:{\displaystyle \varepsilon _{r}}
606:"Ethanol Precipitation of DNA"
604:Zeugin JA, Hartley JL (1985).
521:"Ethanol Precipitation of DNA"
519:Zeugin JA, Hartley JL (1985).
400:can greatly improve recovery.
102:This relation is reflected in
1:
363:strong enough to form stable
631:Crouse J, Amorese D (1987).
555:Crouse J, Amorese D (1987).
457:Phenol–chloroform extraction
718:
76:due to its highly charged
646:(2): 3–5. Archived from
570:(2): 3–5. Archived from
371:or Li plays this role .
160:separated by a distance
472:SCODA DNA purification
384:
328:
228:
201:
174:
154:
127:
69:
697:Laboratory techniques
382:
329:
229:
202:
175:
155:
153:{\displaystyle q_{2}}
128:
126:{\displaystyle q_{1}}
82:"like dissolves like"
63:
17:Ethanol precipitation
244:
211:
184:
164:
137:
110:
442:using isopropanol.
344:together by way of
89:dielectric constant
47:as an antisolvent.
385:
324:
224:
197:
170:
150:
123:
70:
702:Molecular biology
356:out of solution.
322:
286:
236:electric constant
173:{\displaystyle r}
93:charged particles
51:DNA precipitation
41:aqueous solutions
709:
679:
678:
670:
661:
659:
658:
652:
637:
627:
625:
624:
610:
586:
585:
583:
582:
576:
561:
552:
543:
542:
540:
539:
525:
516:
507:
496:
333:
331:
330:
325:
323:
321:
320:
311:
310:
309:
300:
299:
289:
287:
285:
284:
283:
274:
273:
254:
233:
231:
230:
225:
223:
222:
206:
204:
203:
198:
196:
195:
179:
177:
176:
171:
159:
157:
156:
151:
149:
148:
132:
130:
129:
124:
122:
121:
717:
716:
712:
711:
710:
708:
707:
706:
687:
686:
685:
673:
665:
656:
654:
650:
635:
630:
622:
620:
608:
603:
595:
590:
589:
580:
578:
574:
559:
554:
553:
546:
537:
535:
523:
518:
517:
510:
500:Joseph Sambrook
497:
490:
485:
448:
377:
370:
338:hydration shell
312:
301:
291:
290:
275:
265:
258:
242:
241:
214:
209:
208:
187:
182:
181:
162:
161:
140:
135:
134:
113:
108:
107:
66:hydration shell
58:
53:
29:polysaccharides
12:
11:
5:
715:
713:
705:
704:
699:
689:
688:
684:
683:
663:
662:
628:
601:
594:
593:External links
591:
588:
587:
544:
508:
487:
486:
484:
481:
480:
479:
474:
469:
464:
459:
454:
452:DNA extraction
447:
444:
405:centrifugation
398:polyacrylamide
376:
373:
368:
319:
315:
308:
304:
298:
294:
282:
278:
272:
268:
264:
261:
257:
252:
249:
221:
217:
194:
190:
169:
147:
143:
120:
116:
57:
54:
52:
49:
13:
10:
9:
6:
4:
3:
2:
714:
703:
700:
698:
695:
694:
692:
682:
677:
672:
668:
653:on 2009-11-22
649:
645:
641:
634:
629:
618:
614:
607:
602:
600:
597:
596:
592:
577:on 2009-11-22
573:
569:
565:
558:
551:
549:
545:
533:
529:
522:
515:
513:
509:
505:
501:
495:
493:
489:
482:
478:
475:
473:
470:
468:
465:
463:
460:
458:
455:
453:
450:
449:
445:
443:
440:
436:
434:
430:
425:
421:
419:
414:
410:
406:
401:
399:
395:
391:
381:
374:
372:
366:
361:
357:
355:
354:precipitating
349:
347:
343:
339:
334:
317:
313:
306:
302:
296:
292:
280:
276:
270:
266:
262:
259:
255:
250:
247:
239:
237:
219:
215:
192:
188:
167:
145:
141:
118:
114:
105:
104:Coulomb's law
100:
98:
94:
90:
85:
83:
79:
75:
67:
62:
55:
50:
48:
46:
42:
38:
34:
30:
26:
22:
18:
655:. Retrieved
648:the original
643:
639:
621:. Retrieved
616:
612:
579:. Retrieved
572:the original
567:
563:
536:. Retrieved
531:
527:
437:
433:denaturation
426:
422:
417:
402:
386:
358:
350:
335:
240:
101:
86:
71:
16:
15:
467:Salting out
439:Isopropanol
365:ionic bonds
346:ionic bonds
99:or in air.
691:Categories
657:2008-09-10
623:2008-09-10
581:2008-09-10
538:2008-09-10
483:References
462:Salting in
396:or linear
64:The first
43:by adding
37:xyloglucan
413:viscosity
277:ε
267:ε
263:π
216:ε
189:ε
78:phosphate
619:(4): 1–2
534:(4): 1–2
446:See also
394:glycogen
375:Practice
342:crystals
31:such as
681:Biology
360:Ethanol
72:DNA is
45:ethanol
667:Portal
429:buffer
418:pellet
234:is an
97:vacuum
56:Theory
33:pectin
27:, and
651:(PDF)
640:Focus
636:(PDF)
613:Focus
609:(PDF)
575:(PDF)
564:Focus
560:(PDF)
528:Focus
524:(PDF)
74:polar
39:from
390:tRNA
133:and
35:and
238:):
25:DNA
21:RNA
693::
642:.
638:.
615:.
611:.
566:.
562:.
547:^
530:.
526:.
511:^
502:,
491:^
392:,
84:.
23:,
669::
660:.
644:9
626:.
617:7
584:.
568:9
541:.
532:7
409:g
369:4
318:2
314:r
307:2
303:q
297:1
293:q
281:0
271:r
260:4
256:1
251:=
248:F
220:0
193:r
168:r
146:2
142:q
119:1
115:q
Text is available under the Creative Commons Attribution-ShareAlike License. Additional terms may apply.
