IGEM:UNAM/2009/Notebook/Modeling logbook Claudia/2010/09/26

From OpenWetWare

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

Working on LovTAP fused to promoters, BBa_K098991,LuxY and Lumazine:Ligations to backbones pSB3K3 ans pSB1C3

Once the plasmids pSB3K3 and pSB1C3 were correctly digested with the enzymes EcoRI and PstI, I have started the dephosphatation reaction in order to prepare them for ligation with promoters-LovTAP,BBa_K098991(cI regulated promoter+RBS+GFP), LuxY and Lumazine constructions.

  • Dephosphatation mixture
Reactive Quantity
DNA 15μL
Buffer 10X 3μL
Dephosphatase Enzyme 1.5μL
HPLC Up to a final volume of 30 μL of the mixture (10.5μL)

Procedure

1.Incubate the samples at 37°C during 20 min.

2.Incubate the samples at 65°C during 10 min.


Repeated experiment:Working on LovTAP fused to promoters, Lumazine and K098991:Ligations to backbone pSB3K3 and pSB1C3

Due to the first attempt to LovTAP fused to promoters, Lumazine and K098991 to backbones pSB1C3 and pSB3K3 failed, I am going to repeat the experiment.

Ligation Procedure:LovTAP fused to promoters, Lumazine and K098991 with backbones pSB1C3 and pSB3K3

1. Prepare the ligation mixture taking into account the quantity of the DNA insert -LovTAP fused to promoters, Lumazine and K098991- and the receiver DNA - Backbones pSB3K3 and pSB1C3 -. This can be check in the following gels.

Ligations LovTAP+promoters and BBa_K098991 to plasmids pSB3K3 and pSB1C3..Lane1:Ladder.Lane2:Plasmid pSB3K3 (EcoRI/PstI)dephosphatated.Lane3:Plasmid pSB1C3 (EcoRI/PstI)dephosphatated.Lane5:BBa_K098991 (EcoRI/PstI).Lane6:Plasmid LovTAP+J23105 (EcoRI/PstI)Colony 5. Lane7:Plasmid LovTAP+J23102 (EcoRI/PstI) Colony5.Lane8: Plasmid LovTAP+J23117 (EcoRI/PstI) Colony 6. Lane9:Plasmid LovTAP+J23114 (EcoRI/PstI) Colony 4.Lane10:Lumazine Mr.Gene PCR amplified product (EcoRI/PstI).
Ligations LovTAP+promoters and BBa_K098991 to plasmids pSB3K3 and pSB1C3..Lane1:Ladder.Lane2:Plasmid pSB3K3 (EcoRI/PstI)dephosphatated.Lane3:Plasmid pSB1C3 (EcoRI/PstI)dephosphatated.Lane5:BBa_K098991 (EcoRI/PstI).Lane6:Plasmid LovTAP+J23105 (EcoRI/PstI)Colony 5. Lane7:Plasmid LovTAP+J23102 (EcoRI/PstI) Colony5.Lane8: Plasmid LovTAP+J23117 (EcoRI/PstI) Colony 6. Lane9:Plasmid LovTAP+J23114 (EcoRI/PstI) Colony 4.Lane10:Lumazine Mr.Gene PCR amplified product (EcoRI/PstI).
Backbones pSB3K3 and pSB1C3..Lane1:Ladder.Lane2 and 3:Plasmid pSB3K3 (EcoRI/PstI)dephosphatated 1, replica 1 and 2.Lane3 and 4:Plasmid pSB1C3 (EcoRI/PstI)dephosphatated 1 and 2.
Backbones pSB3K3 and pSB1C3..Lane1:Ladder.Lane2 and 3:Plasmid pSB3K3 (EcoRI/PstI)dephosphatated 1, replica 1 and 2.Lane3 and 4:Plasmid pSB1C3 (EcoRI/PstI)dephosphatated 1 and 2.


  • Ligation mixture using plasmid pSB3K3
Reactive Quantity
DNA insert (LovTAP-J23117, LovTAP-J23105, LovTAP-J23114, LovTAP-J23102 and K098991) 6μL LovTAP and promoters/ 4μL K098991
DNA plasmid (pSB3K3) 3μL
Buffer 10X T4 Ligase 2μL
Enzyme T4 Ligase 1μL
HPLC Up to a final volume of 20 μL of the mixture (8μL)
  • Ligation mixture using plasmid pSB1C3
Reactive Quantity
DNA insert (LovTAP-J23117, LovTAP-J23105, LovTAP-J23114, LovTAP-J23102 and Lumazine PCR amplified product from Mr.Gene) 6μL
DNA plasmid (pSB1C3) 3μL
Buffer 10X T4 Ligase 2μL
Enzyme T4 Ligase 1μL
HPLC Up to a final volume of 20 μL of the mixture (8μL)

2. Incubate the sample at 16°C overnight.

3. Transform the cells. Click here for the protocol.

4. Culture the cells in the proper selective medium, in our case LB + Kanamycin.

5. Incubate the petri dishes at 37°C overnight.

6. Re-culture the resultant colonies in the proper selective medium; incubate them at 37°C overnight.

7. Analyze the colonies with Colony PCR to confirm that they contain the correct ligation

The primers that I am using are:

Forward (5'->3'): Preffix primer.

Reverse:(5'->3'): Suffix primer.

These primers would amplify LovTAP ligated to each promoter and BBa_K098991, if the ligation was correctly done.

Getting more sample:Working on LovTAP fused to promoters: Ligation to backbone pSB3K3 for characterization and pSB1C3 for DNA submission

Once the ligations of LovTAP with promoters were confimed, I am going to digest them with EcoRI and PstI again, in order to fuse them to backbones pSB3K3 and pSB1C3.

  • LovTAP + promoters: Ligation to backbones: Restriction enzymes EcoRI and PstI.

Plasmids used:

LovTAP + J23117 isolated form colony 6.

LovTAP + J23105 isolated form colony 5.

LovTAP + J23114 isolated form colony 4.

LovTAP + J23102 isolated form colony 5.

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 20 min.

Results: Restriction enzyme assay Preparing LovTAP and promoters for plasmids backbone ligations

According to the next image, it seems that all the LovTAP ligations with promoters, were correctly digested because the bands obtained are around the expected size of the LovTAP gene (889nt) plus the promoters lenght.

LovTAP + promoters: Restriction enzyme assay with EcoRI and PstI. Lane1:Ladder.Lane9:LovTAP+J23114 colony 4.Lane10:Plasmid LovTAP+J23117 colony 6. Lane11:Plasmid LovTAP+J23105 colony 5.Lane12:Plasmid LovTAP+J23102 colony 5.The other lanes are samples from other experiments.
LovTAP + promoters: Restriction enzyme assay with EcoRI and PstI. Lane1:Ladder.Lane9:LovTAP+J23114 colony 4.Lane10:Plasmid LovTAP+J23117 colony 6. Lane11:Plasmid LovTAP+J23105 colony 5.Lane12:Plasmid LovTAP+J23102 colony 5.The other lanes are samples from other experiments.

These digestions will be fused to plasmids pSB3K3 and pSB1C3.



Personal tools