can anyone help me to explain it? - (Mar/30/2007 )
I cut my vector first with NheI O/N, and then add some 5M NaCl in it, to adjust the buffer to the same as bamH1 buffer, and incubate with BamH1 for 2 hours. When I run the gel, I can know that my vector is cut and looks fine after NheI. And after BamH1, I can see two bands, one is my cut vector , the other one is the insert dropped off . But the wierd thing is , my vector should be like 10ug, and the gel looks like just 1 ug, How can I explain it. Where has my plasmid gone???
Could be star activity of the Bam. Check the pH of your NaCl. If it's significantly basic that could promote star activity (no idea how it would get that way - would have to be contamination, but you never know).
Any smear or sign of DNA degradation that might have occured? One should use the right amount of enzyme to DNA and cut the reaction time to avoid such events.
Run a similar amount of DNA on gel to confirm the actual DNA. Dont rely only on spectrophotometer reading for DNA conc.
I totally agree with scolix. Sometimes, the reading from Nanodrop is not accurate, especially the gel extraction product.
