10xLigationBuffer:0.5MTris-HClpH7.8,50mMMgCl2,0.1Mb-mercaptoethanol,50mMATP,5mg/mlBSA Asusuallywhenpipettingenzymereactionskeepthetubesoniceduringallmanipulations.Cip-treatadigestedvectorcutwithonlyasingleenzyme.Ifthevectorwasdouble-digested:phenolizepriortoligating.Useequimolaramountsofvectorandinsert.Asaruleweuse50ngvector(3kb)and50nginsert(3kb).Morevaluesaregiveninthetablebelow.Recalculateaccordinglywhenusingdifferent-lengthvectors.Don"tuselessthan8ngorsoforfragmentssmallerthan0.5kb.InthecaseofsmallfragmentscircularizationoccursandremovestheFfromtherxn.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:
FUsedinStandardLigation vector(50ng,3kb) LengthofF(kb) AmountofF(ng) 0.5 8.3 1 16.7 1.5 25 2 33.3 2.5 41.7 3 50 3.5 58 4 66.7 5 83.3 6 100 7 116.3 Materials:
Reagent/Tool Supplier Cat.-# 
BSA ATP T4DNALigase
