Solution 3/3

A. Digest vector pQE-60with NcoI [C^CATGG] (113) and HindIII [A^AGCTT] (152).

5'--TTA.AC/C.ATG.GGA.GGA.TCC.AGA.---.CAC.TA/A.GCT. TAA.TTA.GCT.GAG--3'
3'--AAT.TG G.TAC/CCT.CCT.AGG.TCT.---.GTG.AT T.CGA./ATT.AAT.CGA.CTC--5'

Resulting fragments following this pQE-60 digestion:

1. 3392 bp HindIII 152 - NcoI 113
     2.     39 bp NcoI      113 - HindIII 152

B. Digest insert pGEM-GOI with NcoI [C^CATGG] (2919) and HindIII [A^AGCTT] (3266).

5'--GGA.TC/C.ATG. GTT.CTT.CCA.GCT.---.TAG.A/AG.CT T.ATC.GAT.TTC.--3'
3'--CCT.AG G.TAC./CAA.GAA.GGT.CGA.---.ATC.T TC.GA/A.TAG.CTA.AAG.--5'

Resulting fragments following this pGEM-GOI digestion:

1. 2995 bp HindIII 3266 - NcoI 2919
2.  347 bp NcoI 2919 - HindIII 3266

Remembe that NcoI restriction site in pGEM-GOI was formed only following the insertion of GOI into pGEM.

Let's examine this solution in more detail. Look at the table below:


Insert
pGEM-GOI
Vector
pQE60

Fill the following table
(class discussion)

Element
pQE60-GOI will contain
pQE60-GOI will NOT contain
Restriction Sites (NsiI,SacI,BamHI,NcoI,BglII,HindIII,ClaI, SmaI,KpnI,EcoRI,XhoI,XbaI,SphI,AatII,ApaI)
Note that some of the Restriction Enzymes cut both plasmids !
    
Poly His
    
Antibiotics Resistance
(Amp, Kan, Neo, Tet)
    

Examine the next solution