A typical digestion requires 1ug DNA for a 50uL reaction which results in DNA that is 20ng/uL. Once I do a cleanup the concentration is too dilute for NGS (or 20ng/uL. I am using genomic DNA.
I think it is possible.
You can also refer to guidelines by NEB:
https://www.neb.com/tools-and-resources/usage-guidelines/optimizing-restriction-endonuclease-reactions
David's answer is quite correct, there is no problem with scale up, I've done digestions at 0.5 to 1ml volumes as well. You can also increase the concentration of DNA, the limiting factor is glycerol concentration as David points out. You can also ethanol precipitate your DNA afterwards instead of a spin column which may not do much to increase concentration.
OK...so there's a few things to consider there. Very appreciative indeed. I am finding the spin columns useless as they are reducing my DNA significantly in the end ....will look at magnetic beads to and do a test against ethanol precipitation. Had never heard of dilution denaturation either! thank you both.
Nowadays all the enzymes from NEB come with buffers already containing BSA. It´s the X.1 and CutSmart nomenclature where they omitted DTT (was only in there for historical reasons they claim) and add BSA for customer convenience. Drawback is, that you always have to re-freeze the buffers since they are less stable now.
The short answer is yes, you can scale up the and keeping the ratios. It appears your problem is DNA recovery not digestion. Spin columns are over sold to you and ethanol ppt is efficient but sometime you have to get rid of the enzyme and other ingredients. Definition of units is important. Amount of enzyme needed is calculated based on number of sites need to be cut. The Units are defined on reference DNA, frequently Lambda DNA, and it usually has limited number of sites for certain enzymes. e.g. 48k lambda has 7 hind-iii sites, if you are cutting a 3 k with 2 sites you need 6 folds per ug and that can get high very fast depending on the enzyme, you get the picture.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Biological Techniques