IGEM:UNAM/2009/Notebook/Modeling logbook Claudia/2010/09/14

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LovTAP-Promoters to plasmid pSB3K3/pSB1C3: Confirmation by enzyme restriction assay

I continue working on LovTAP-Promoters ligations to plasmid pSB3K3/pSB1C3. 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 plasmids pSB3K3/pSB1C3 . So that, if I cut with this enzyme the PCR products amplified of each colony, 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.

PCR amplified products used:

LovTAP-J23105 + plasmid pSB3K3 from colonies 6 and 7.

LovTAP-J23114 + plasmid pSB3K3 from colony 3.

LovTAP from Mr. Gene + plasmid pSB1C3 from colonies 2, 6 and 10.

Restriction enzyme assay:Testing LovTAP and promoters ligations

  • Single restriction reaction: Mixure

Restriction enzyme: SalI.

Reactive Quantity
PCR amplified DNA 5μL
Buffer 3 2μL
Enzyme 1μL.
HPLC Up to a final volume of 20 μL of the mixture (11μL)

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 pSB1C3/pSB3K3 ligations

According to the next image, it seems that only the LovTAP (Mr.Gene) ligations with plasmid pSB1C3, that were tested are true positives, because in lanes 6,7 and 8 there is a band around 500nt, which is the expected fragment cutting with SalI because the SalI recognition site is at the middle of the LovTAP gene (889nt). The band pattern obtained from three of the four positive controls was as expected.

Confirmation:LovTAP and pSB1C3/pSB3K3 ligations. Lane1:Ladder.Lane2: (Control +)Plasmid LovTAP+J23117 colony 6 digested with SalI/PstI.Lane3: (Control +)Plasmid LovTAP+J23105 colony 5 digested with SalI/PstI.Lane4: (Control +) PCR amplified product LovTAP+J23117 colony 6 digested with SalI.Lane5: (Control +) PCR amplified product LovTAP+J23105 colony 5 digested with SalI. All these samples correspond to previous LovTAP ligations with J23 promoters. Lane6, 7 and 8:Colony PCR LovTAP (Mr.Gene) amplified product fused to plasmid pSB1C3. Colonies 10,2 and 6 respectively.Lane9: Colony PCR LovTAP-J23114 amplified product fused to plasmid pSB3K3 from colony 3. Lane 10 and 11:Colony PCR LovTAP-J23105 amplified product fused to plasmid pSB3K3 from colony 6 and 7 respectively.