10xLigationBuffer:0.5MTris-HClpH7.8,50mMMgCl2,0.1Mb-mercaptoethanol,50mMATP,5mg/mlBSA QuickprepphageDNAgetspreparedaccordingtoourstandardprotocol.WeuseLamBDaDASHorEMBL4lambdaphagesasvectors.ThustheinsertgetsreleasedusingEcoRI.BeawarethattheinsertusuallycarriesinternalEcoRIsitesaswell.EcoRIDigestion 
10xEcoRIbuffer 0.5ml phageDNA(200ng) 4.5ml EcoRI(20u/ml) 0.2ml total 5.2ml LigationMix(2x) 10xligasebuffer 1.0ml digestedvector(0.1mg/ml) 1.0ml H2O 6.0ml total 8.0ml LigationRxn Insert Control ligationmix 4.0ml 4.0ml insert 1.0ml ---ml T4DNAligase(400u/ml) 0.2ml 0.2ml Total 5.2ml 4.2ml Solutions:
LigationBuffer(10x) 1MTris-HClpH7.8 500.0ml 1MMgCl2 50.0ml b-mercaptoethanol 7.0ml 100mMATP 50.0ml 0.1g/mlBSA 50.0ml H2O 343.0ml Total 1ml Remarks:
Materials:
Reagent/Tool Supplier Cat.-# 
BSA ATP T4DNALigase
