Hi all,
I use Q5 NEB high-fidelity polymerase for PCR. I usually do 4 replicates, and ethanol precipitate the PCR products (0.1 sample volume 3 M NaAc, 2 sample volume of ethanol). I got a big white precipitate at the bottom of my tube after centrifugation. Re-dissolve the pellet in water and re-do the ethanol precipitation does not help much. Does anyone know what is the precipitate? Is that all my DNA, or does salts also precipitate? Can I use it for downstream experiments, such as restriction digestion? Thank you!
Hello, use sodium acetate 4M solution 50ul in 1ml sample keep in cold 2-4*C for 10min than centrifuge and use ethanol purification.
I cannot see what the q5 buffer is but 2 possibilities are 1 you are using the high GC buffer which almost certainly contains 5M betaine and some DMSO so you do have a lot of salt present.
2 the information sheet ( attached) does say that the buffer may precipitate normally in the first few uses so there are probably salts or organic compounds which are not very soluble in water present.
I would try not re precipitating but just adding 70%ethanol to the precipitate and re centrifuging in the hope that much of the salt will redissolve. If the salt is betaine then most enzymes will work in its presence but you cannot be certain
If you intend to do further downstream processing I would strongly recommend to purify the PCR with a silica-based cleanup column instead of bothering with ethanol precipitation. And if you insist on ethanol precipitation, why not include a deproteinization step with saturated phenol first?
- It sounds like something has precipitated out of the PCR buffer although I am surprised it cannot be re solubilised
- Regardless repeat PCR with new Taq buffer aliquot etc
Hello, use sodium acetate 4M solution 50ul in 1ml sample keep in cold 2-4*C for 10min than centrifuge and use ethanol purification.
Hi everyone:
Thanks for the answer. I have been using Qigen PCR purification kit to purify my PCR products. Its fast and works great.
- Badges
- Science method