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                 2 Experiment
 
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                2.1 Method
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                 <p>After sequencing and ensuring the sequence was correct, we applied it to the experiments. Store at 4℃.
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                 <p>The two ends of the pcDNA3.1(+)-3×HA-Gal4-VP64-NLS linearized vectror need to be complementary to the two ends of the corresponding recombinant fragments to ensure the success of homologous recombination ligation.
 
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Revision as of 15:26, 8 October 2023


pcDNA3.1(+)-3xHA-Gal4-KRAB-NLS linearized vector

The purpose of the linear vector is to perform homologous recombination with the KRAB homologous recombination fragment to obtain the target product pcDNA3.1 (+) -3xHA-Gal 4-KRAB-NLS plasmid.

pcDNA3.1(+)-3×HA-GAL4-VP64-NLS

We based on the sequence of the pcDNA3.1 (+) plasmid.The Gal 4 sequence, 3×HA sequence were linked into the pcDNA3.1 (+) plasmid with the help of homologous recombination experiments. Special primers were designed and then the plasmid was cut into linear sequences of about 5600bp by PCR.The two ends of this linear sequence can be complementary to the two ends of the KRAB homologous recombination fragment , and the purpose is to conduct homologous recombination with the KRAB homologous recombination fragment to obtain the target product pcDNA3.1 (+) -3 HA-Gal 4-KRAB-NLS plasmid.

1 Pattern Diagram


Fig.1 The agarose gel electrophoresis of pcDNA3.1(+)-3×HA-Gal4-KRAB-NLS linearized vector

2 Experiment

Based on the sequence of the pcDNA3.1 (+) plasmid, With the help of the GAL 4 homologous recombination fragments and homologous recombination experiments.We designed and synthesized the target product.

3.Caution

The two ends of the pcDNA3.1(+)-3×HA-Gal4-VP64-NLS linearized vectror need to be complementary to the two ends of the corresponding recombinant fragments to ensure the success of homologous recombination ligation.