Cloning of DWF4
Professor,
- Yeaterday I transfer to agar rice samples for repeating the lamina bending with JMJ703 plants and phyB mutants. With the JMJ703 plants I will repeat the experiments with the same conditions that I've been using. In case of phyB, after the response of Prof. Park I will grow them in white light conditions.
- As I mention too I wanted to continue the cloning of BIN2, and as you also suggest me just clone that product from the last time. But last weak after I obtain more bands in the PCR I did gel extraction and purification, and that product I amplify it again... obtaining just one band, that was the band that I wanted to sequence yesterday and you suggest just to proceed cloning. But before that I did pcr purification of that band, and when I load the product of the purification I saw again a lot of non specific band, which I can not explain the reason.... So I am now thinking in two obtions. One is just to proceed cloning with the product of the gel purification. The second is to amplify again the product of the gel purification by PCR and check again that there is no other band and do the cloning with that PCR product.
- Also I have other primers for DWF4 cloning and after gradient PCR I obtained a product with my expected size, but the band is weak, so I will repeat it with cDNA of plants treated with pcz to have a little more of DWF4 product and also I am trying to adjust the PCR conditions for the same purpose.
That is the plan for today...
Claudia
- Yeaterday I transfer to agar rice samples for repeating the lamina bending with JMJ703 plants and phyB mutants. With the JMJ703 plants I will repeat the experiments with the same conditions that I've been using. In case of phyB, after the response of Prof. Park I will grow them in white light conditions.
- As I mention too I wanted to continue the cloning of BIN2, and as you also suggest me just clone that product from the last time. But last weak after I obtain more bands in the PCR I did gel extraction and purification, and that product I amplify it again... obtaining just one band, that was the band that I wanted to sequence yesterday and you suggest just to proceed cloning. But before that I did pcr purification of that band, and when I load the product of the purification I saw again a lot of non specific band, which I can not explain the reason.... So I am now thinking in two obtions. One is just to proceed cloning with the product of the gel purification. The second is to amplify again the product of the gel purification by PCR and check again that there is no other band and do the cloning with that PCR product.
- Also I have other primers for DWF4 cloning and after gradient PCR I obtained a product with my expected size, but the band is weak, so I will repeat it with cDNA of plants treated with pcz to have a little more of DWF4 product and also I am trying to adjust the PCR conditions for the same purpose.
That is the plan for today...
Claudia