1. Preparealigationmix:

   LigationMix(2x)
   10xligasebuffer	1.0ml
   digestedvector(0.1mg/ml)	1.0ml
   H2O	6.0ml
   total	8.0ml

2. divideligationmixintotwoEppendorftubes

   LigationRxn
   	Insert	Control
   ligationmix	4.0ml	4.0ml
   insert	1.0ml	---ml
   T4DNAligase(400u/ml)	0.2ml	0.2ml
   Total	5.2ml	4.2ml

3. incubatefor2-3h(orovn)at14°C
4. proceedwiththetransformationoftheappropriateE.colistrain

Solutions:

10xLigationBuffer:0.5MTris-HClpH7.8,50mMMgCl2,0.1Mb-mercaptoethanol,50mMATP,5mg/mlBSA 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:

Asusuallywhenpipettingenzymereactionskeepthetubesoniceduringallmanipulations.Cip-treatadigestedvectorcutwithonlyasingleenzyme.Ifthevectorwasdouble-digested:phenolizepriortoligating.Useequimolaramountsofvectorandinsert.Asaruleweuse50ngvector(3kb)and50nginsert(3kb).Morevaluesaregiveninthetablebelow.Recalculateaccordinglywhenusingdifferent-lengthvectors.Don"tuselessthan8ngorsoforfragmentssmallerthan0.5kb.InthecaseofsmallfragmentscircularizationoccursandremovestheFfromtherxn.

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

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