IGEM:UNAM/2009/Notebook/Modeling logbook Claudia/2010/08/25

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Working on LovTAP and promoters ligations

Once LovTAP from colony 1 was correctly digested and extracted from gel, Daniela and I have started the ligation protocol in order to join LovTAP with the previously chosen promoters: J23102, J23105, J23106, J23110, J23114, J23116, J23117, which plasmids were digested and dephosphatated.

Ligation Procedure:LovTAP + Promoters

1. Prepare the ligation mixture taking into account the quantity of the DNA insert -LovTAP- and the receiver DNA -plasmids harboring the promoters-. This can be check in the following gel.

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 + Ampicillin.

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, if the ligation was correctly done. The expected size of the product is approximately 915 nt, considering the lenght of LovTAP 889 nt and the sizes of the promoters and the primers.

Results ligation: LovTAP + Promoters

We decided to focus our effort in the ligations of LovTAP with two weak promoters: J23117 and J23114, one medium promoter: J23105 and one strong promoter J23102.

I chose two colonies from each ligation.

Promoter + LovTAP Colonies
J23117 3 and 6
J23114 4 and 7
J23105 5 and 7
J23102 3 and 5

The resuls for each colony PCR are showed in the next image. It seems that LovTAP+J23117 from colony 6 was succesfully ligated; colony 5 and 7 harboring LovTAP+J23105, colony 4 harboring LovTAP+J23114 and colony 5 harboring LovTAP+J23102 (not showed in this gel), were succesfully ligated, too. This is because the amplified product in each colony is around the expected size 915 nt (889nt for LovTAP plus promoter lenght), however we still have to confirm that the insert is LovTAP sequence, beacause these amplified products might be false positives, if RFP gene (887 nt) was re-ligated to each promoter, considering that the yield of the dephosphatation reaction is not 100%.

The plasmids from the aforementioned colonies were isolated with the protocol previously described.

I have to look for a enzyme restriction site unique for LovTAP sequence that does not exist in RFP gene nor in the plasmid J61002.

LovTAP + promoters ligations. Lane1:Ladder. Lane9:LovTAP+J23117 colony 3.Lane10:LovTAP+J23117 colony 6.Lane11:LovTAP+J23105 colony 5.Lane12:LovTAP+J23105 colony 7.Lane13:LovTAP+J23114 colony 7.Lane14:LovTAP+J23114 colony 4.Lane15:LovTAP+J23102 colony 3.The other lanes are samples from other experiments

Working on LovTAP, LuxY and CcaS/CcaR and LuxAB: Edinburgh Shipment

Once the PCR reactions for LovTAP and CcaS/CcaR gene were successfully done, I purified the PCR mixures using theHigh Pure PCR Product Purification kit from Roche. As well, I extracted LuxY from gel using the previously PCR amplified fragments. In order to confirm that the amplified products are still conserved in the new solutions and that the the LuxY extraction from gel was correctly done, I ran a gel. The results are showed in the next image.

Edinburgh Shipment:LovTAP, CcaS/CcaR, LuxAB and LuxY. Lane1:Ladder. Lane2:LovTAP PCR cleaned sample 1.Lane3: LovTAP PCR cleaned sample 2.Lane4: CcaS/CcaR PCR cleaned.Lane 5: LuxAB extracted from Vibrio fischeri PCR cleaned.Lane6: LuxY sample 1 extracted from gel.Lane7:LuxY sample 2 extracted from gel.Lane8:LuxY sample 3 extracted from gel.The other lanes are samples from other experiments

The PCR purifications for LovTAP(lanes 2&3) and CcaS/CcaR (lane4) were correctly done, the fragments obtained are around the expected sizes: 889 nt for LovTAP and 3204 nt for CcaS/CcaR. The LuxAB PCR purification(Lane 5) was also correctly done, the band obtained is around the expected size 2148 nt (2091nt for LuxAB genes plus 24 and 33 nucleotides added at each end with the enzyme restriction recognition sites), but it still has the PCR forward and reverse primers-the strong band at the bottom- because their lenght is 68 and 58 nucleotides nt, respectively.

Finally, the LuxY extraction from gel (lanes 6, 7 and 8) was correctly done, beacause only one band was obtained from each sample. The band obtained is around the LuxY expected size (820nt); sample 1 and 2 have more LuxY DNA than sample3, thus they will be used for future analysis and Edinburgh shipment.