I have been triying to recover plasmid from filter paper send by Addgene, using TE buffer and only water. However, in both cases there's no evidence from DNA when I run an electrophoresis gel. Also, I took 5 ul to transform competent cells and doesn´t work.
Further, I tried to incubate 10min/65°C with buffer TE and water and i obtain the same result. The plasmid has been storage for less than one week to 4°C and the delivery only took 2 days. I'll appreciate if somebody can to suggest a protocol step by step.
Regards
don't expect to see the DNA in a gel (there is not enough amount), but transforming E. coli with the TE soaking the paper should work. the only alternative i can think of is trying to PCR the plasmid useing the TE with the paper as template (with phosphorylated primers) and ligate and transform the PCR product...
good luck!
How much water/buffer did you use in the first extraction? I have performed similar DNA extractions from filter paper from addgene in the past, and I try to use as little water as possible (~50ul at most, also depending on the size of the paper/how much weight of DNA they sent you) in the hopes to not overly dilute my sample prior to transformation. Since you may have diluted your DNA sample with the repeated extraction, I would combine every aqueous extraction you have done, combine it with the paper yet again, heat it back up, vortex it vigorously (trying to get every little piece of DNA off of the paper that you possible can), and then vacufuge your sample to concentrate it back down to maybe ~20ul or less. Hopefully at this point your sample will have a concentration high enough to get a successful transformant. Best of luck.
Max
Thanks all for your answers. Yesterday, I tried a protocol similar to the recomendation of Max Levine and finally it works.
Regards
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