Hi researchers,
Now I'm sequencing a 8kb PCR product from patient sample. I'm doing gel purification after PCR to get my product. One difficulty in this procedure is that I need to do several reactions for each sample to get enough PCR product. Then if I do gel purification, the work is overwhelming(I need to prepare over 100 samples).
I heard that beads can be used to purify PCR product and based on different ratio, certain length of DNA can be reclaimed. Can anyone give me some suggestion about which beads I can use?(I have AMpure beads in my lab.) And if I want to reclaim the 8kb PCR product, which ratio should I use?
Thanks so much!
Hi,
As per my experience most of these kits will give less or less yield as the size of the product reaches close to 10 kb. The best approach will be to optimize your PCR to get more yield before going for purification
bye
if you are Sanger sequencing why not either use 2 nested primers just inside your long amplicon. They do not have to be great primers because the genome target is now small. Alternatively nested primers at 1kb intervals and re amplify and sequence. This is more pcr but less purification.
You should get more yield from your PCR (increase number of cycle) with 100 us and then purification.
I am not sure of the exact volume, but can suggest one way to figure out the right volume. Use diluted DNA ladder (Kb plus or one that has band of the size of your interest) and test out the different bead volumes that can purify the size of your interest. If you don't have non-specific bands on your gel, things will be easier/cleaner.
- Badges
- Science method