I have a plasmid resuspended in TE and i have to digest it, do i have to do an ethanol precipitation first?
Besides that, should i resuspend my DNA pellets in TE, Tris 10 mM or Roux if i need to digest them? cause all my downstream apps require digestions.
Thanks.
Martin.
Probably not if your DNA is quite concentrated. If you only need to digest 1uL of your sample, it will be diluted down 50X in a final reaction mix of 50uL and won't interfere with the enzyme. The enzyme buffers always have a large excess of Mg2+ as well.
I might start to worry if your DNA is so dilute that you'd need to add something like 20uL to your reaction because then you'd have a large amount of carryover EDTA. You could just add a bit more enzyme if this is the case though
Probably not if your DNA is quite concentrated. If you only need to digest 1uL of your sample, it will be diluted down 50X in a final reaction mix of 50uL and won't interfere with the enzyme. The enzyme buffers always have a large excess of Mg2+ as well.
I might start to worry if your DNA is so dilute that you'd need to add something like 20uL to your reaction because then you'd have a large amount of carryover EDTA. You could just add a bit more enzyme if this is the case though
I agree with Govinda, if your DNA solution is concentrated enough you can skip EtOH precipitation.
I recommend to resuspend your DNA pellets in TE buffer (10mM Tris; 0.1 mM EDTA; pH 8.0). You can also use TE to resuspend oligonucleotides.
Well, many labs including us, used to resuspend the final DNA from plasmid or genomic DNA in nuclease free water instead of TE buffer to avoid EDTA which could interfere during sequencing steps.
If you are only planning for digestion and cloning of your DNA product, you can simply resuspend in TE buffer as suggested by Govinda or Christian. You don't require to go another ethanol precipitation as you may lose your DNA. Make sure your DNA should be of high concentration (100ng/ul) with high purity. Set up digestion reaction in 20ul volume with 2-3 ul of your DNA sample. It works well.
All the best.
TE is avoided if the down-stream experiment involves DNA sequencing, as requested by the sequencing technician.
Drop Dialysis can be used to decrease the amounts of EDTA as well as salts and other small molecules from DNA samples.
Marusyk, R. and A. Sergeant (1980) A Simple Method for Dialysis of Small-Volume Samples. Analytical Biochemistry 105: 403-4.
Saraswat, M., Grand, R.S., and W.M. Patrick (2013) Desalting DNA by Drop Dialysis Increases Library Size upon Transformation. Biosci. Biotechnol. Biochem. 77: 402-4.
Dear Martin,
I agree with Hung King Tiong, If you need DNA sequencing downstream application, TE should be avoided. Instead use the Ultrapure H20.
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