IGEM:Slovenia HS 2015/2009/Notebook/Test/2015/04/22

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Gene isolation

  • We have ordered specific oligonucleotides (BdhA, CtfAB).Our goal was the isolation of genes reffering to Clostridium acetobutylicum. There is a table beneath. As oligonucleotides we have used BdhA (R), Bdha (F), ctfAB (R), ctfAB (F), 16S (F), 16S (R). Basically we were changing only the source of isolation.

Isolation sources:

  • broth
  • 10x diluted broth
  • DNA
  • 100x diluted DNA


Mixture for PCR reaction (master mix)

component quantity (µL)
oligo 1 (10µM) 1 10
oligo 2 (10µM) 1 10
dNTP (2mM) 1 10
MgCl2 2 20
buffer 2 20
dH2O 11 110
Vtot. / 190

Electrophoresis

The next step was Agarose gel electrophoresis. We can see that there are some of the products, but they do not correspond of all the masses of the products. CfAB and BdhA are both 1000 bp long. 16S is 600 bp long. The reaction have to be repeated.

Agarose electrophoresis: 1-broth, 2-100x diluted broth, 3-DNA, 4-100x diluted DNA


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