software to replace Vector NTI and a simple cloning problem - (Jan/04/2009 )
We are thinking about replacing Vector NTI with other competing software because of recent price increase of the Vector NTI. Any recommendation on which comperting software we should buy? Price less than $1500 per software for perpetual license over 5 years would be reasonable.
Second question that we look for answer is that our simple cloning hasn't worked yet. This is two sticky ends (Hind III and EcoR I) ligation: about 2.8kb insert ligated into a 7.1kb binary vector. The insert was a product of partial digestion and then eluted from TAE (modified) gel and then recovered using DNA gel-recovery kit of column. Backbone was cut and then either used directly for ligation or gel-purified. The ligation ratio of insert vs. backbone has been 3:1, 4:1, and 5:1, but no colonies have developed.
The performer of this cloning job is an experinced cloner and ligase/buffer has been from Fermantus first and then from NEB. The DNA for the ligation are from Quegen midipre with high quality. Each enzymatic cut was checked for a complete cut and the unit of restriction enzyme vs DNA and reaction volumes were preperly set to avoid star activity.
Any thought/suggestion will be geratly appreciated.
Have a great new year!
I don't use vector NTI...so can't comment on that.
Some clonings are tricky and take time. Concentration of the insert and vector are critical and one of the ratios should work. My suggestion would be to screen multiple colonies at one go. Just streak each colony on a plate and pool 3 colonies in one inoculation tube. This way you can screen 72 colonies in 24 minipreps and whereever you see the required bands, go back to the plate and screen each colony independently. I faced a problem cloning 6kb insert in 12kb vector, took a lot of time and finally i got one colony. My job was done.
Also, if you can avoid running the fragments on gel, it helps. Somehow running on the agarose gel decreases ligation efficiency.
Hope it helps.