IGEM:UNAM/2009/Notebook/Modeling logbook Claudia/2010/08/21

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Working on LuxY synthesized plasmid from Mr.Gene

In order to test the correct lenght of LuxY synthesized, I am going to do a PCR reaction using as template the plasmid harboring LuxY from Mr.Gene.

Primers:

Forward:Preffix primer

Reverse:Suffix primer


PCR Template:

Plasmid harboring LuxY from Mr.Gene


Reactive Quantity
MgCl2 2.5μL
Buffer 10X Taq 5μL
dNTP's 5μL (0.4mM)
Primer Forward 3μL (5pmol/μL)
Primer Reverse 3μL (5pmol/μL)
DNA template (Mr.Gene Plasmid:LuxY 2μL + 8μL HPLC) 2μL
Taq polymerase 1μL
HPLC Up to a final volume of 50 μL of the mixture

Working on LovTAP promoters:Dephosphatation of strong,medium and weak promoters for ligation

Once the plasmids harboring the seven promoters were correctly digested with the enzymes SpeI and PstI, I have started the dephosphatation reaction for each one in order to prepare them for ligation.


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 (11.5μL)


Procedure

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

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


Results: Dephosphatation of strong, medium and weak promoters for ligation with LovTAP

Dephosphatation of promoters. Lane1:Ladder. Lane2: J23102. Lane3: J23105.Lane4: J23106.Lane5: J23110. Lane6: J23114.Lane7: J23116.Lane8: J23117.The other lanes are samples from other experiments
Dephosphatation of promoters. Lane1:Ladder. Lane2: J23102. Lane3: J23105.Lane4: J23106.Lane5: J23110. Lane6: J23114.Lane7: J23116.Lane8: J23117.The other lanes are samples from other experiments

Working on LovTAP: Extraction from gel band

In order to extract LovTAP from the gel ready for ligation with J23 promoters and plasmid pSB1C3, I'm going to use the gel extraction Kit from QIAGEN.

The extraction of LovTAP from gel was decided considering the results previously reported, which are found in the next link.

Gel extraction protocol

1. Prepare the gel using low melt agarose at 1%.

  • 1g agarosoe
  • 100 mL TAE

2. Run the gel in cold room.

3. Follow the gel extraction protocol from QIAGEN


  • Electrophoresis parameters:
Voltage Running Time
80 50min

The samples used to run the gel were:

  • LovTAP plasmid from colony 1 digested with XbaI and PstI.
  • LovTAP plasmid from colony 1 digested with EcoRI and PstI.

These were obtained previously.

Resuts: LovTAP Gel extraction protocol

We were only able to extract LovTAP from colony 1 digested with XbaI and PstI, the sample digested with EcoRI and PstI was lost during the gel loading step.

After the extraction we ran the sample in a new gel, in order to know if the gel extraction was done correctly. In lane 6 of the next image I loaded LovTAP extracted from colony 1 and digested with XbaI and PstI. There is a band around the expected size (889nt) for LovTAP, but there is another weak band down LovTAP, thus the LovTAP extaction from gel has a little contamination from other digested product. Despite this contamination we decided to continue with the ligation procedure in order to join LovTAP with the choosen promoters. So that, I also loaded the plasmids harboring the promoters that were digested and dephosphated for ligation -Lane 7 to Lane 13- to calculate the quantity of the samples that must be used for ligation.

Dephosphatation of promoters & LovTAP from colony 1 extracted from gel band. Lane1:Ladder. Lane 2: Plasmid 17. Lane3: J23100.Lane4: LRE XbaI/PstI.Lane5: LRE EcoRI/PstI. Lane6: LovTAP colony extracted.Lane7: J23102.Lane8: J23105.Lane9: J23106.Lane10: J23110.Lane11: J23114.Lane12: J23116.Lane13: J23117.Lane14: LuxAB PCR cleaned. The other lanes are samples from other experiments
Dephosphatation of promoters & LovTAP from colony 1 extracted from gel band. Lane1:Ladder. Lane 2: Plasmid 17. Lane3: J23100.Lane4: LRE XbaI/PstI.Lane5: LRE EcoRI/PstI. Lane6: LovTAP colony extracted.Lane7: J23102.Lane8: J23105.Lane9: J23106.Lane10: J23110.Lane11: J23114.Lane12: J23116.Lane13: J23117.Lane14: LuxAB PCR cleaned. The other lanes are samples from other experiments




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