Alkalinelysisplasmidminiprep

BasedonCurrentProtocolsinMolecularBiologyprotocol
SamanthaOrchard
Materials:
Glucose/Tris/EDTA(GTE)solution
For100ml:
4.5ml20%glucose
2.5ml1MTrisCl,pH8.0
2ml0.5MEDTA
To100mlwithwater
Autoclaveandstoreat4C
Recommended:addRNasetoworkingaliquot(e.g.10mlGTE+100lRNase(10mg/ml))and
storeat4C
NaOH/SDSsolution(makefresh)
For1ml:

For10ml:

200l
2ml
1NNaOH
100l
1ml
10%SDS
700l
7ml
Water
Potassiumacetatesolution
For100ml:
29.5mlglacialaceticacid
pHto4.8withKOHpellets(takesalotandgetshotmayboil)
Addwaterto100ml(mayhelptoaddabitofwaterwhileaddingKOH)
Storeatroomtemperature
10mg/mlDNasefreeRNase
Protocol:
Pellet1.5mlcellculture
Resuspendpelletin100lGTE+1lRNase(or100lpremixedGTE/RNasemixture)
Add200lfreshNaOH/SDSsolution;mixbyinverting~8times
Add150lpotassiumacetatesolution;mixbyinverting~8times
Spin1min
Transfersupernatenttofreshtube
Add800l100%ethanol;letsit2minatroomtemperature
Spin1min
Washpelletwith1ml70%ethanol
Drypellet
Resuspendpelletin30l10mMTrisClpH(8or8.5)orTEorwater