I'm a doctoral student with early experiences with recombinant protein synthesis. Since I still have to start my experiment and I do not yet know the quantities of DNA and plasmid that I will have to work with to get a good reaction yield, I ask you for advice on the quantity of DNA, plasmid and restriction enzyme I should use. I've learned that the amount of RE used depends on how much DNA and plasmid is used. Furthermore, there are precise relationships between the quantities of these two reagents. But what's the right amount of DNA to start with?
Does anyone have any helpful tips to help me figure out how to set these steps? Thank you so much
Hi,
You can refer to this: https://www.neb.com/en/faqs/2012/09/13/how-should-i-set-up-a-restriction-digest.
I hope this will useful for you. Good luck!
If you are performing manual RE digestion individually (i.e RE, buffer, plasmid DNA, water in 50µl reaction) then use 1.5-3µg plasmid DNA.
if you going through golden gate ligation 70-100ng plamid DNA needed (PCR based ligation).
It depends upon what you are intending to do. If you just want to look at bands on a gel, then a few hundred ng of plasmid is sufficient. If you are going to do further manipulations or gel purify a band, then you will want more DNA, maybe a few micrograms.
- Badges
- Science topic
- Similar topics
- Psychology