g EcoRI a nd chew back a 3 overhang eg PstI Alternatively the Quick Blunting Kit from New England Biolabs httpwwwnebcomnebecommproductsintlproductE1201asp is optimized to blunt and phosphorylate DNA ends for cloning in less than 30 minutes Klenow fro ID: 62482
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AlexAitken Page 1 11/10/2012 Blunting ofDNA PreparedbyMsAlexAitken Insomeinstancestheendsoftheinsertorvectorrequireblunting . PCRwithaproofreadingpolymerasewillleaveapredominantlybluntend . T4DNAPolymeraseorKlenowwillfillina5´overhang(e.g.,EcoRI)a nd chewbacka3´ overhang(e.g.,PstI) . Alternatively,theQuickBluntingKit fromNewEnglandBiolabs http://www.neb.com/nebecomm/products_intl/productE1201.asp isoptimized to blunt and phosphorylateDNAendsforcloninginlessthan30minutes . KlenowfromNewEnglandBiolabs KlenowFragment(3´ - )isnotsuitableforgeneratingbluntendsbecause itlacksthe3´ - templated3´a dditions. YoumustusetheLargeKlenowFragment. Protocolforbluntingends by3'overhangremovaland3'recessedendfill - in 1. DNAshouldbedissolvedin1XNEBuffer1 - 4orT4DNALigaseReaction Buffer 2. Supplement with33MeachdNTP. 3. Add1unitKl enowpermicrogramDNA 4. Incubate 15minutesat25°C. 5. StopreactionbyaddingEDTAtoafinalconcentrationof10mMandheating at75°Cfor20minutes. 6. AsEDTAisnowpresentthisshouldbegelextractedbeforeproceedingto ligations. T4DNAPolymerase fromNewEnglandBiolabs Bestenzymeforcreatingbluntends Protocolforbluntingends by3´ overhangremovaland3´recessedendfill - in 1. DNAshouldbedissolvedinany1XrestrictionenzymereactionNEBuffer 2. Supplemented with100µMdNTPs. 3. Add1unit T4DNA PolymerasepermicrogramDNA 4. Incubate 15 minutesat12°C. 5. StopreactionbyaddingEDTAtoafinalconcentrationof10 mMandheating to75°Cfor20 minutes. 6. AsEDTAisnowpresentthisshouldbegelextractedbeforeproceedingto ligations. Note : Elevatedtemperatures,excessiveamountsofenzyme,failuretosupplement withdNTPsorlongreactiontimesmayre sultinrecessedendsduetothe3' exonucleaseactivityoftheenzyme.