I'm trying transform the construct-FLAG ripk3 - under the selection of Amp/puro, which is present on filter paper at 180 ŋg/µl. i had by dissolved it in 10µl TE buffer and stored at 4°c for one day. from that I've used 2µl with 100 µl DH5α for transformation. I've repeated this experiment for 4 times, But I'm unable to get successful transformation. can you please suggest me the better way of transformation and suggest me regarding concentrations of the DNA also. and tell me the better way to retain DNA from filter paper at high concentration
First check to be sure your competent cells are still good by doing a positive plasmid control. Also the amp selection is for bacteria, the puromycin for eukaryotes. So you should probably just use the ampicillin selection for your initial transformants in E. coli.
Hi Rajan,
1. First, you have to make sure the exact spot of the plasmid on the filter membrane. Did they circle the spot area for you? Because after the plasmid dry up, you cannot tell where the exact location of the spot.
2. How big is the filter you tried to isolate the plasmid out? I think that 10 uL TE you use was too little. You can see the article provided by Tata Santosh Rama Bhadra Rao. The article suggests using 100 uL TE. We use about the same TE amount too.
3. Then, make sure the competent cells are good (as mentioned by Michael), and the selection antibiotic is correct.
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