should I go on to the ligation? urgent - (Mar/25/2007 )
Hi everyone.
I'm trying to clone an 500bp insert into pet28b vector by using Nhe1 and BamH1 as restriction enzyme. First i digested the vector and insert by Nhe1 overnight. This morning, I did BamH1 digestion for four hours after the PCR purification. But when I see the gel result for this digestion. I found both my vector and insert were degraded. But I still have some left running on the right positions for both vector and insert. I've cut the gel and put them in the 4 degree. I wonder in this case can I continue to the ligation step? the amount is not that much for the vector. Too sad right now.
I also run some sample after the Nhe1 digestion for both vector and insert, and they are fine. What would cause the degradation for bamH1 step??
Many thanks
youyou
I found using BamHI if you cut for too long time, it will have this problem. I usually cut the vector for 30min, and PCR product for 1 hour (because the amount is larger than vector). I think 4 hours is too long for it.
I am not sure whether you can go for the ligation step, but I will give it a try. And at the same time, you can try to cut the material again with shorter time.
I'm trying to clone an 500bp insert into pet28b vector by using Nhe1 and BamH1 as restriction enzyme. First i digested the vector and insert by Nhe1 overnight. This morning, I did BamH1 digestion for four hours after the PCR purification. But when I see the gel result for this digestion. I found both my vector and insert were degraded. But I still have some left running on the right positions for both vector and insert. I've cut the gel and put them in the 4 degree. I wonder in this case can I continue to the ligation step? the amount is not that much for the vector. Too sad right now.
I also run some sample after the Nhe1 digestion for both vector and insert, and they are fine. What would cause the degradation for bamH1 step??
Many thanks
youyou
thanks a lot. I'll try it. 
I am not sure whether you can go for the ligation step, but I will give it a try. And at the same time, you can try to cut the material again with shorter time.
I'm trying to clone an 500bp insert into pet28b vector by using Nhe1 and BamH1 as restriction enzyme. First i digested the vector and insert by Nhe1 overnight. This morning, I did BamH1 digestion for four hours after the PCR purification. But when I see the gel result for this digestion. I found both my vector and insert were degraded. But I still have some left running on the right positions for both vector and insert. I've cut the gel and put them in the 4 degree. I wonder in this case can I continue to the ligation step? the amount is not that much for the vector. Too sad right now.
I also run some sample after the Nhe1 digestion for both vector and insert, and they are fine. What would cause the degradation for bamH1 step??
Many thanks
youyou
another question, previously BamH1's buffer is always BamH1 buffer, why this time the new enzyme i ordered came in with the NEbuffer 3?? They are not using the specific buffer for BamH1 any more? May this be the problem??
And how much enzyme should I used for 1ug DNA incubating 1 hour to avoid the star activity?
If you have both insert and vector of the correct size, you might as well try the ligation expt. If it works, you're a hero ligation expert; if it doesn't, it was a long shot, anyway!
If the ligation doesn't work, may I suggest you cut down your BamHI digestion. Look at the definition of a unit: 1 ug of lambda (which has quite a few BamHI sites in each molecule) in 1 hr digestion. Unless you have a huge amount of DNA and very little enzyme, you are overdigesting by a long way (although, by 4 hrs, most of the enzyme is dead). Bam is also one of those enzymes that is very sensitive to its conditions. Use only what the data sheet recommends (1 units or 2 should be all you need, especially for a plasmid with only a single Bam site), don't go over 10% volume as enzyme, and keep the incubation time to 1 hr.
Happy ligating / digesting / re-ligating