Team:Goettingen/Notebook/evolution August

gat and aroA* amplification

09.08.18

PCR was performed in order to amplify the DNA fragments aroA* and gat. The g Block DNA ordered from <a href="https://eu.idtdna.com/pages">Integrated DNA Technologies</a> serves as template DNA for the PCRs of aroA* and gat genes. The PCR samples were compounded as described in the <a href="https://2018.igem.org/Team:Goettingen/Experiments">Experiments</a> section. Approximately about 50 ng of the template DNA was used. 

       <img src="T--Goettingen--GAT%26AroA.png">

PCR products of gat and aroA* genes after gel electrophoresis

PCR-products were purified and and digested with the restriction enzymes PstI and EcoRI. After incubation of both digestion samples with aroA* and gat for 30 minutes at 37 °C, the samples were cleaned up using the same kit again. After that, concentrations of the digested genes has been determined via nanodrop.

Ligation of digested gat and aroA* into pBQ200

10.08.18

In order to ligate each of the two genes into pBQ200 the following samples were prepared.

µl Sample Substance
13,5 µL H2O
2 µL T4-Buffer
2,5 µL pBQ200 (Vector)
1 µL aroA*
1 µL T4-Ligase
µl Sample Substance
13,1 µL H2O
2 µL T4-Buffer
2,5 µL pBQ200 (Vector)
1,4 µL gat
1 µL T4-Ligase

Additionally, one sample without insert was prepared which serves as a religation control. Ligation and religation samples were incubated for 2 hours at room temperature. After incubation competent DH5α cells were transformed with the ligated samples.

Test digest of pBQ200 with aroA* and gat

14.08.18

Positive clones were isolated and an over night culture of them was used for plasmid isolation. After that, the isolated plasmids were digested using the restriction enzymes PstI and EcoRI.

               <img src="T--Goettingen--AroATestverdau.png" alt="AroA-Test digest ">

               <img src="T--Goettingen--GATTestverdau.png " alt="Gat-Test digest ">

The test digest of the plasmid pBQ200 with aroA*

This picture shows the test digest of pBQ200 with gat

After that the sequences of each plasmid with either gat or aroA* was checked via sequencing.