1. digestgenomicphageDNA:

   EcoRIDigestion
   10xEcoRIbuffer	0.5ml
   phageDNA(200ng)	4.5ml
   EcoRI(20u/ml)	0.2ml
   total	5.2ml

2. incubatefor15minat37°C(heatingblockorwaterbath)
3. heatfor5minat65°C(heatingblock)
4. add2ml5MNH₄Acand10mlisopropanol
5. spinfor20minat4°C
6. washpelletwith70%EtOH(-20°C),speedvacanddissolvepelletin4ml0.1xTE(ca50ng/ml)
7. preparealigationmix:

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

8. divideligationmixbetweentwoEppendorftubes

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

9. incubatefor2-3h(orovn)at14°C
10. 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:

QuickprepphageDNAgetspreparedaccordingtoourstandardprotocol.WeuseLamBDaDASHorEMBL4lambdaphagesasvectors.ThustheinsertgetsreleasedusingEcoRI.BeawarethattheinsertusuallycarriesinternalEcoRIsitesaswell.

Materials:

Reagent/Tool	Supplier	Cat.-#
BSA
ATP
T4DNALigase

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