Enzyme restriction - (Aug/18/2011 )

Hi every one
I have ligated my insert to the PENTR /D TOPO vector and trying hard to get my insert band after enzyme digestion.. Its really surprising for me that when i digest my plasmid with ASCI and NOt I enzyme , i cannot see the positive band for my insert but the vector size that is 2580 bp looks ok , my pro is that if i digest the same plasmid with different enzyme like ASCI and Xbai ,in that case i got my required insert but the vector size become very high may be more than 5000 bp... i tried this many time and got same result now i am stuck????? What to do i have no idea ??

if some body have same experience plz do share with me or any suggestion ...
Thanks
Pcr product size 1530 bp
Vector size 2580 bp

waiting for reply

Your next step should be to do some single enzyme digests of your vector with each of those enzymes. That, or pick a different clone (or several) from your transformation plate and analyze those.

Thanks for your suggestion, i already tried several clones from my plates... now i will try single enzyme digestion but in that case can i see my insert band??

Single enzyme digests may show you the length of your plasmid. Since it appears that the double digests produce inconsistent results on the plasmid length, this should tell you what is really happening. I'm guessing you have a doubled insert lacking one of the cut sites you anticipate. If you run uncut plasmid on the gel, you can also determine if there are enzymes which don't cut at all.

Better, though, would be to find a clone with the right properties.

If your goal is only to verify the insert presence in the vector you may as well do a colony PCR. When it comes to the restriction enzyme digest, do you do a AscI & NotI double digest or consecutive AscI followed by clean up and NotI digest? AscI/NotI double digest is very unlikely to work in any NEB buffer as the buffer preferences are quite incompatible, while if you do AscI/XbaI the optimal buffer is the same. I do not understand how the treatment of the same sample with the different pairs of enzymes would lead to different vector size though.

Cheers

yes i just want to confirm the prescence of my insert only ,i do AscI overnight then NOt1 .. i have done colony PCR by using M13 primers btu they also show the same result for all the samples thats why its difficult for me to pick the correct colony having insert???? yes this is also surprising for me why the vector size is different
Anyways thakns for your suggestion

Hi , Phage434
i tried with single enzyme digestion but the size of plasmid is very big .
my required size is around 4000 bp as 2580 vector + 1530 insert but the result is around 7000 bp???
i dont know whats going wrong do you have any idea ???

This is kind of what I expected, and why I wanted you to do this. I think your plasmid has multiple copies of portions of the plasmid. Do you care about the plasmid portion, or do you only care about the insert?

My main　focus is on insert only ???