Hi
After purified my digested plasmid from agarose gel, i used Nano-drop which gave me 33.1ng/ul. After that, I used Gibson to insert my gene and then run PCR 50c for 1/h.I got nothing after transformation .When I go back to check the concentration of my digested plasmid by using agarose gel because I thought that Nano-drop gave me wrong result I found that no plasmid there, it disappeared can anyone help me, why my digested plasmid disappeared?
Hi Marym,
Just to clarify your procedure. You purified your digested plasmid from your gel, and then quantified it to 33ng/uL. Then you performed a ligation and transformation, isolated that construct using a plasmid prep, then did PCR and got no results? You then went back and checked your initial digested vector by running it on a gel and saw nothing?
A couple points: 50 cycles is way on the high end, especially doing PCR from plasmid. If you can't get a glowingly strong product after 35 cycles, you might have to troubleshoot your PCR profile.
How much of your digested vector did you run on the gel? I typically run at least 100-500ng on a gel to ensure I see it (but I have old eyes haha).
Did you go back and check the concentration of your initial vector using the nanodrop again? What was the result?
Barry
Hi Barry
The ligation step by using Gibson Assembly requires incubation at 50°C for 1/h or run PCR one cycle 50 °C, 1/h, which I did. Then did transformation but I got no colonies in the plate. so I checked my initial digested plasmid by using agarose gel but the plasmid just disappeared.
I run 200 ng of digested plasmid in agarose gel, I found no band no plasmid. I did not check the concentration of my plasmid by nanodrop I just used agarose gel to find that.
It sounds like your plasmid prep failed. Did you try running out some of your purified plasmid at the beginning of your experiment? 33 ng/ul is a very, very low concentration for purified plasmid.
Start over with a new plasmid prep and run out a couple micrometers of your product on an agarose gel. You can't rush through these steps.
- Badges
- Science method