Difference between revisions of "Team:TU Darmstadt/Notebook/sec1/K1602004"

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     <figcaption><b>Figure 1</b> PCR of GRE3 (X). The size of the amplified product was around 1.0 kbp. DNA marker: 2-Log DNA Ladder (NEB).</figcaption>
 
     <figcaption><b>Figure 1</b> PCR of GRE3 (X). The size of the amplified product was around 1.0 kbp. DNA marker: 2-Log DNA Ladder (NEB).</figcaption>

Revision as of 15:47, 16 September 2015

K1602004 - aldose reductase (GRE3)


Figure 1 PCR of GRE3 (X). The size of the amplified product was around 1.0 kbp. DNA marker: 2-Log DNA Ladder (NEB).
The GRE3-gene was synthesized by GeneArt and amplified through PCR using the GRE3 (FW) and GRE3 (REV) oligonucleotides The PCR product was verified by agarose gel electrophoresis and subsequently purified out of the gel because of unknown amplificates in the range of a few hundred kbp. The purified GRE3 amplicon was cut with EcoRI and PstI and ligated into a pSB1C3 vector using T4-ligase. The ligation product was transformed into E.coli Top 10 by heat shock transformation and the cells were spread out on a CMP agar plate. Clones were screened with colony PCR using VF2 and VR oligonucleotides. Plasmid DNA was isolated from positive clones which were verified by sanger sequencing.