alkaline lysis sambrook

8/16/2019 Alkaline Lysis Sambrook

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Alkaline Lysis sambbrook (English)

1. Inoculate 2 ml of rich medium (LB, !, or !errific Broth) containing the a""ro"riate

antibiotic #ith a single colony of transformed bacteria. Incubate the culture o$ernight

at %&' #ith $igorous shaking

!o ensure that the culture is adeuately aerated*

!he $olume of the culture tube should be at least four times greater than the $olume

of bacterial culture.

!he tube should be loosely ca""ed

!he culture should be incubated #ith $igorous agitation.

2. +our 1. ml of the culture into a microfuge tube. entrifuge at ma-imum s"eed for %

seconds at /' in a microfuge. 0tore the unused "ortion of the original culture at /'.

%. hen entrifugation is com"lete, emo$e the medium by as"iration, lea$ing the

bacterial "ellet as dry as "ossible.

!his ste" can be con$eniently accom"lished #ith a dis"osable "i"ette ti" or +asteur

"i"ette attached to a $acuum line and side arm flask ("lease see figure 13&). 4se a

gentle $acuum and touch the ti" to the surface of the liuid. 5ee" the ti" as far a#ay

from the bacterial "ellet as "ossible as the fluid is #ithdra#n from the tube. !his

minimi6es the risk that the "ellet #ill be sucked into the side arm flask. Alternati$ely,

remo$e the su"ernatant using a "i"ettor or +asteur "i"ette and bulb. 4se the "i"ette ti"

to $acuum the #alls of the tube to remo$e any adherent dro"lets of fluid.


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!he "enalty for failing to remo$e all traces of medium from the bacterial "ellet is a

"re"aration of "lasmid 78A that is resistant to clea$age by restriction en6ymes. !his

is because cell #all com"onents in the medium inhibit the action of many restriction

en6ymes. !his "roblem can be a$oided by resus"ending the bacterial "ellet in ice cold

0!E (.2 - $olume of the original bacterial culture) and centrifuging again.

Lysis of ells

/. esus"end the bacterial "ellet in 1 9l of ice3cold Alkaline lysis solution I by

$igorous $orte-ing.

:ake sure that the bacterial "ellet is com"letely dis"ersed in Alkaline Lysis 0olution

I. $orte-ing t#o microfuge tubes simultaneously #ith their bases touching increases

the rate and efficiency #ith #hich the bacterial "ellets are resus"ended.

!he original "rotocol (Birnboim and 7oly, 1;&;) called for the use of Lyso6yme at

this "oint to assist in dissolution of the bacterial cell #alls. !his ste" can be safely

omitted #hen dealing #ith bacterial cultures of less than 1 mL in $olume.

. Add 2 9l of freshly "re"ared Alkaline lysis solution II to each bacterial sus"ension.

lose the tube tightly, and mi- the contents by in$erting the tube ra"idly fi$e times.

Do not vortex! 0tore the tube on ice.

:ake sure that the entire surface of the tube comes in contact #ith Alkaline Lysis

0olution II.


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&. entrifuge the bacterial lysate at ma-imum s"eed for minutes at /' in a microfuge.

!ransfer the su"ernatant to a fresh tube.

=. (Optional ) Add an eual $olume of "henol*chloroform. :i- the organic and aueous

"hases by $orte-ing and then centrifuge the emulsion at ma-imum s"eed for 2

minutes at /' in a microfuge. !ransfer the aueous u""er layer to a fresh tube.

0ome in$estigators find the e-traction #ith "henol*choloroform to be unnecessary.

>o#e$er the elimination of this ste" sometimes results in 78A that is resistant to

clea$age by restriction en6ymes. !he "ur"ose of e-tracting #ith cholorofom is to

remo$e residual "henol from the aueous "hase. +henol is slightly soluble in >2?, but

it can be dis"laced into the organic "hase by choloroform. ears ago, it #as common

"ractice in some laboratories to detect residual "henol in 78A "re"arations by smell.

!his "ractice is no longer recommended.

;. +reci"itate nucleic acids from the su"ernatant by adding 2 $olumes of ethanol at room

tem"erature. :i- the solution by $orte-ing and then allo# the mi-ture to stand for 2

minutes at room tem"erature.

1. ollect the "reci"itated nucleic acids by centrifugation at ma-imum s"eed for

minutes at /' in a microfuge.

It is best to get into the habitof al#ays arranging the microfuge tubes in the same #ay

in the microfuge rotor, i.e., in order, #ith their "lastic hinges al#ays "ointing out#ard.

!he "reci"itate #ill collect on the inside surface furthest from the center of rotation.

5no#ing #here to look makes it easier to find $isibles "reci"itates and to dissol$e

@in$isible "reci"itates efficiently. Labeling both the sides and to"s of tubes "ro$ides

clear identification of each tube, e$en if the ink smudges.

11. emo$e the su"ernatant by gentle as"iration as described in 0te" % abo$e. 0tand the

tube in an in$erted "osition on a "a"er to#el to allo# all of the fluid to drain a#ay.


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4se a 5im#i"e or dis"osable "i"ette ti" to remo$e any dro"s of fluid adhering to the

#alls of the tube.

12. Add 1 ml of & ethanol to the "ellet and in$ert the closed tube se$eral times.

eco$er the 78A by centrifugation at ma-imum s"eed for 2 minutes at /' in a

microfuge.

1%. emo$e all of the su"ernatant by gentle as"iration as described in 0te" %.!ake care

#ith this ste", as the "ellet sometimes does not adhere tightly to the tube.

1/. emo$e any beads of ethanol that form on the sides of the tube. 0tore the o"en tube at

room tem"erature until the ethanol has e$a"orated and no fluid is $isible in the tube

(31 minutes).

If the "ellet of 78A is dried in a desiccator or under $acuum. It becomes difficult to

dissol$e under some circumstances and may denature (0$aren et al., 1;=&). 7rying

the "ellet for 131 minutes at room tem"erature is usually sufficient for the ethanol

to e$a"orate #ithout the 78A becoming dehydrated.

1. 7issol$e the nucleic acids in 9l of !E ("> =.) containing 2 9gCml 78ase3free

8ase A ("ancreatic 8ase). Dorte- the solution gently for a fe# seconds. 0tore the

78A solution at 32'.

or recommendations on troubleshooting, "lease see table 13 in "rotocol %.

alkaline lysis sambrook

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