Hello,I have a problem with restriction analysis of ligation product. I have cloned BMP-2 gene into pUET3 vector. Then, I've tried to choose restriction enzymes with NEBcutter, this is my ligation product: http://tools.neb.com/NEBcutter2/cutshow.php?name=912b6cbb- I have to choose enzymes not affected by methylation. I've found only one 2cutter that meets this condition (NspI), but it gives 2593 bp and 693 bp products. I'm worried that the difference is too big and I won't see smaller product on agarose gel. The same problem occurs with 3cutters. How can I solve this problem? I would be grateful for any help
You should be able to see 2.6 and 0.7kb products on a 0.7% agarose gel run at 120V for 20mins.