IGEM:UNAM/2009/Notebook/Modeling logbook Claudia/2010/09/02

From OpenWetWare

Jump to: navigation, search
UNAM-Genomics-Mexico team Main project page
Previous entry      Next entry

Working on cI inverter construction, pSB3K3 backbone and E0240 part

Once the plasmids harboring the parts showed in the next table were correctly isolated, I am going to digest some with EcoRI and PstI and the others with XbaI and PstI in order to fuse them with their corresponding part.

Part Lenght
K098991 (cI regulated promoter+RBS+GFP) 933 bp
P0451(RBS+cI repressor) 930bp
E0240 (RBS+GFP protein) 876 bp
J04450 pSB3K3 backbone 1010 bp
  • Restriction enzymes EcoRI and PstI:

K098991 preparation for ligation with plasmid pSB3K3 and J04450 preparation for ligation with promoters-LovTAP, trpL-cI inverter and trpL-GFP constructions for characterization.

Plasmids used:

Plasmid harboring K098991 isolated from colony 8.

Plasmid harboring J04450 isolated from colony 8. Two replicas

Reactive Quantity
Plasmid DNA 15μL
Buffer 2 4μL
BSA 1μL
Enzymes 2μL for each one.
HPLC Up to a final volume of 40 μL of the mixture (16μL)


  • Restriction enzymes XbaI and PstI:

P0451 and E0240 preparation for ligation with constitutive promoter (J23101) and trpL promoter

Plasmids used:

Plasmid harboring P0451 isolated from colony 6.

Plasmid harboring E0240 isolated from colony 5. Two replicas

Reactive Quantity
Plasmid DNA 15μL
Buffer 2 4μL
BSA 1μL
Enzymes 2μL for each one.
HPLC Up to a final volume of 40 μL of the mixture


The reactions were incubated at 37°C overnight.

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

Results restriction enzyme assays

Restriction enzyme assays: EcoRI/PstI and XbaI/PstI. Lane1:Ladder. Lane2:J4450 colony 8 replica 1 (EcoRI/PstI). Lane3:J4450 colony 4 replica 2 (EcoRI/PstI).Lane4: E0240 colony 5 replica 1 (XbaI/PstI). Lane5: E0240 colony 5 replica 2 (XbaI/PstI).Lane6:P0451 colony 6 (XbaI/PstI) .Lane7: K098991 colony 8 (EcoRI/PstI).
Restriction enzyme assays: EcoRI/PstI and XbaI/PstI. Lane1:Ladder. Lane2:J4450 colony 8 replica 1 (EcoRI/PstI). Lane3:J4450 colony 4 replica 2 (EcoRI/PstI).Lane4: E0240 colony 5 replica 1 (XbaI/PstI). Lane5: E0240 colony 5 replica 2 (XbaI/PstI).Lane6:P0451 colony 6 (XbaI/PstI) .Lane7: K098991 colony 8 (EcoRI/PstI).

The sizes of the digested products are around the expected lenght for each part.



Personal tools