Ligating 2 fragments of equal size... - any tricks? (Jan/05/2009 )
Hey everyone and Happy New Year! 
I am going to help out our research assistant with some cloning as she's having difficulties. My cloning always works (touch wood) and so she asked me for help. It sounds like basic cloning to me but perhaps I am missing something and that's why it's not working for her??
Basically I need to ligate BamHl-INSERT-Not1 (5kb) into a vector (5.3kb) with BamHI/Not1. Are there any issues ligating two pieces of fairly easy size?
Just thought I would ask the experts before I get my clone on
Thanks in advance!
Clare
the only immediate problem that i can see is the NotI site being too close to the BamHI site in the vector. NotI requires at least 8bp skirting the restriction site to cut with any efficiency.
Else, it looks pretty much like any normal ligation.
I am going to help out our research assistant with some cloning as she's having difficulties. My cloning always works (touch wood) and so she asked me for help. It sounds like basic cloning to me but perhaps I am missing something and that's why it's not working for her??
Basically I need to ligate BamHl-INSERT-Not1 (5kb) into a vector (5.3kb) with BamHI/Not1. Are there any issues ligating two pieces of fairly easy size?
Just thought I would ask the experts before I get my clone on
Thanks in advance!
Clare
Nopy dopy!! You can clone 10kb insert in 3kb vector too. In BAC vectors - 500Kb insert in 10Kb vector. In YAC vectors..
Else, it looks pretty much like any normal ligation.
wOOt! The BamHI and Not1 sites are not too close to eachother.
Wish me luck
Clare