PLASMID ISOLATION FROM E.

Coli (ALKALINE LYSIS METHOD)

REAGENTS AND SOLUTIONS
Glucose/Tris/EDTA (GTE) solutio
50 mM glucose
25 mM Tris Cl, pH 8.0
10 mM EDTA
Autoclave and store at !C
N!OH/SDS solutio
0.2 " "a#H
1$ %&t'vol( sodium dodec)l sul*ate %+D+(
,repare immediatel) -e*ore use
" M #ot!ssiu$ !cet!te solutio% #H &'(
2..5 ml glacial acetic acid
/#H pellets to pH .8 %several(
H2# to 100 ml
+tore at room temperature %do not autoclave(
1. 0noculate 5 ml sterile 12 medium &it3 a single -acterial colon). 4ro& to saturation
%overnig3t(.
2. +pin 1.5 ml o* cells 20 sec in a microcentri*uge at ma5imum speed to pellet. 6emove
t3e supernatant &it3 a ,asteur pipet.
T3e spins in steps 2 and 7 can -e per*ormed at !C or at room temperature. 1onger
spins ma8e it di**icult to resuspend cells.
9. 6esuspend pellet in 100 :l 4TE solution and let sit 5 min at room temperature. 2e
sure cells are completel) resuspended.
. Add 200 :l "a#H'+D+ solution, mi5 -) tapping tu-e &it3 *inger, and place on ice *or
5 min.
5. Add 150 :l potassium acetate solution and vorte5 at ma5imum speed *or 2 sec to mi5.
,lace on ice *or 5 min. 2e sure mi5ing is complete.
7. +pin 9 min as in step 2 to pellet cell de-ris and c3romosomal D"A.
;. Trans*er supernatant to a *res3 tu-e, mi5 it &it3 0.8 ml o* .5$ et3anol, and let sit 2
min at room temperature to precipitate nucleic acid
8. +pin 1 min at room temperature to pellet plasmid D"A and 6"A.
.. 6emove supernatant, &as3 t3e pellet &it3 1 ml o* ;0$ et3anol, and dr) pellet under
vacuum.
10. 6esuspend t3e pellet in 90 :l TE -u**er and store as in support protocol. <se 2.5 to
5 :l o* t3e resuspended D"A *or a restriction digest.
Contaminating 6"A ma) inter*ere &it3 detection o* D"A *ragments on t3e agarose gel=
it can -e destro)ed -) adding 1 l o* a 10 mg'ml 6"ase solution %D"ase>*ree( to t3e
digestion mi5ture.