After freeze, squeeze, and centrifugation of an agarose gel slice containing a PCR fragment of 250 to 600 bp, can you use the supernatant containing the DNA directly for sequencing? I'm thinking that phenol: chloroform would not be necessary because I don't have any proteins in there that need to be precipitated. Then, I can use ethanol precipitation directly which would allow me to concentrate the DNA but I might loose some as well so in the end it might not be optimal or necessary (low amount of DNA required for sequencing). I also have a LOT of samples to process and I am wondering if I can't just used the supernatant obtained after freeze-squeeze & centrifugation straight away for sequencing (which would be in TAE buffer). Has anyone done that before ? I will compare the yields experimentally next week, but any insight is welcome.
I would expect trouble with the carryover of salts and EtBr from the electrophoresis buffer, and even low-level nuclease contamination. But I've never done it so please, do report back on the results!
Thanks for your reply Alejandro. I thought about the salts issue but some DNA purification kit elutes in TE so I'm thinking there shouldn't be major problems from this end. I will let you know.
Hi Alejandro,
Yes, the sequencing worked, the bigger the band the cleaner the sequencing, no big surprise there.
- Badges
- Science method
- Similar topics
- Biological Science
- Genetics
- Genetic Analysis
- genetic techniques
- Sequencing