IGEM:UNAM/2009/Notebook/Modeling logbook Claudia/2010/08/26

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Working on LovTAP and promoters ligations: Confirmation by enzyme restriction assay

I continue working on LovTAP ligations with the promoters J23117, J23105, J23114 and J23102. I selected SalI restriction enzyme, in order to confirm LovTAP ligations, because there is a recognition site for that enzyme inside LovTAP coding region, approximately at the middle of the gene. The SalI recognition site is not present in RFP gene nor in plasmid J61002. So that, if I cut with this enzyme the plasmids extracted harboring the four aforementioned ligations, I will confirm that they are true positives (the insert is LovTAP and not RFP gene).

I am going to do a single restriction reaction using SalI enzyme, and a double restriction reaction using SalI and PstI enzymes.

Plasmids used:

LovTAP + J23117 isolated from colony 6.

LovTAP + J23105 isolated from colony 5 and 7.

LovTAP + J23114 isolated from colony 4.

LovTAP + J23102 isolated from colony 5.

Negative control: Plasmid J61002 harboring J23110 promoter.

Restriction enzyme assay:Testing LovTAP and promoters ligations

  • Single restriction reaction: Mixture

Restriction enzyme: SalI.

Reactive Quantity
Plasmid DNA 5μL
Buffer 3 2μL
BSA 1μL
Enzyme 1μL.
HPLC Up to a final volume of 20 μL of the mixture
  • Double restriction reaction: Mixure

Restriction enzymes: SalI and PstI.

Reactive Quantity
Plasmid DNA 5μL
Buffer 3 2μL
BSA 1μL
Enzymes 1μL for each one.
HPLC Up to a final volume of 20 μL of the mixture

The restriction reactions were incubated at 37°C overnight.

Note: Inactivate the enzymes at 80°C during 10 min.

Results: Restriction enzyme assay Testing LovTAP and promoters ligations

According to the next image, it seems that all the LovTAP ligations with promoters, that were tested are true positives, because in lanes 5, 7, 9, 11 and 13, where samples digested with SalI and PstI were loaded, there is a weak band around 400 and 500nt, which is the expected fragment cutting with SalI and PstI, because the SalI recognition site is at the middle of the LovTAP gene (889nt) and the PstI recognition site is at the 3’ end. The three bands obtained at lanes 5 and 11, might be produced for partial restriction reactions.

In lanes 4,6,8,10 and 12, samples digested with SalI were loaded. The respective plasmid is now linearized, thus there is only one band in each lane.

Confirmation:LovTAP + promoters ligations. Lane1:Ladder.Lane2: Plasmid J61002 harboring promoter J23110 digested with SalI. Lane3: Plasmid J61002 harboring promoter J23110 digested with SalI and PstI.Lane4: Plasmid LovTAP+J23105 colony 7 digested with SalI.Lane5:Plasmid LovTAP+J23105 colony 7 digested with SalI and PstI.Lane6: Plasmid LovTAP+J23102 colony 5 digested with SalI.Lane7:Plasmid LovTAP+J23102 colony 5 digested with SalI and PstI.Lane8: Plasmid LovTAP+J23114 colony 4 digested with SalI.Lane9:Plasmid LovTAP+J23114 colony 4 digested with SalI and PstI.Lane10: Plasmid LovTAP+J23117 colony 6 digested with SalI.Lane11:Plasmid LovTAP+J23117 colony 4 digested with SalI and PstI.Lane12: Plasmid LovTAP+J23105 colony 5 digested with SalI.Lane13:Plasmid LovTAP+J23105 colony 5 digested with SalI and PstI.Lane14: Plasmid J61002 harboring promoter J23110.Lane15:Plasmid LovTAP+J23102 colony 5.