two notI sites, only want one cut - (Mar/31/2006 )
HI,
My vector contains two NotI sites at 1 and 2600bp. i only want one cut, and insert a polylinker in it and use sequencing to select cutting at 1 bp. I tried different time points, but still get complete digestion. How can I get one cut product?
Thanks
do you have time to do site-directed mutagenesis on your vector and get rid of the extra NotI site?
that is, if you cannot get partial-digestion to work...that would have been my first choice; by the way, what all have you tried that it will only cut both times??
2 min, 5 min? reduced temperature and enzyme amount?
HI,My vector contains two NotI sites at 1 and 2600bp. i only want one cut, and insert a polylinker in it and use sequencing to select cutting at 1 bp. I tried different time points, but still get complete digestion. How can I get one cut product?
Thanks
Hi zheng791,
You could try to digest the DNA with less enzyme, for example, 0.2U and incubate for 10 minutes. Stop the reaction by heating, and run a gel to determine the adequate amount of enzyme and incubation time.
Hi thanks for your suggestions.
As it is a shuttle vector only for cloning, the two notI sites are used to put down half of the vector and connect the insersion half on the expression vector,.The notI sites must be kept. I will try lower temperatures and less enzyme.
By the way, there are several buffers to choose. Buffer 2 is 50% activity, 3 is 100% and 4 is 25% activity. Does the low activity buffer works for partial digestion?
hi
what i've done once is to do a pcr of the vector. Primers are in oposite sense as classical. I PCR-amplified the vector.
Primers are holding the extra polylinker, and i added a new unique site.
Digestion of pcr product (digest only it as the template do not contain it) and ligation.
Minipreps shows up the relevant colony.
Alternative : do a gel purification of the pcr product, insert it in TOPO vector, and then cut the topo by unique ensyme site added by pcr, purify fragment, ligation and screening.
OR, you could mix the plasmid with EtBr. As the plasmid is supercoiled, it will not take up the EtBr. When the NotI is added, it will cut once and the EtBr immediately inserts, thereby stopping anymore cuts. Clean up the EtBr and proceed with your cloning. My colleague says it works a treat for cutting just once in supercoiled DNA.
Fiona.