Colony selection - (Sep/29/2005 )
Insert digested with NheI and XbaI site (both are compatible cohesive ends) is ligated into a vector with XbaI single cut. How to select the exact colonies express ligation only? Coz single cut control also grow some colonies.
Blue/white screening may help you.
Treat your vector with calf alkaline phosphatase (CIP) before ligation.
Dear SmallGTPase,
if blue-white selection not applicable and you didn't dephosphorylate your vector, grow up about 12 colonies (before inoculating into the culture medium, streak a tiny bit of each bacterial clone onto a clean plate - Master Plate) and do a mini-prep, then screen by restriction digest. Takes 2 days to do and then you'll know what you have and can also sequence from the mini-preped DNA if you want to be doubly sure you have what you want.
In addition to the suggestions above,if your insert was gotten by PCR amplification.you can extract plasmid DNA of recombinant colonies,and detect the colonies by PCR using the original primers.if the recombinant plasmid was tranformmed into cells, you could get the insert again.
Or, to save even more time (and money), screen by colony PCR using a bit of the colony directly into the PCR reaction. This saves growing an overnight culture and extracting the plasmid.
Further, if you have a primer directed against your vector and one against your insert, you can screen for the presence of and orientation of the insert at the same time (if orientation is important).
We do colony routinely for screening transformants...
Homebrew is offering a great solution, if you already have primers for the plasmid.
Note: if you have sequencing primers where one sits in the vector and one sits on your insert, and the difference in Tm is less than 5˚C, use these to screen. You can detect presence of insert and orientation. Thus, you do not require extra PCR primers :-)
the insert is around 3kb and the vector aound 5.5kb. my inserts are directly cut from other plasmids.
after extracting plasmid DNA from mini-cultures, multiple bands were detected. the inappropriate bands (much smaller but not exact vector size) are much brighter than the appropriate band (~8-9kb). I cut these weak "appropriate bands" and retransformed into competent cells. unfortunately get no colony.
after extracting plasmid DNA from mini-cultures, multiple bands were detected. the inappropriate bands (much smaller but not exact vector size) are much brighter than the appropriate band (~8-9kb). I cut these weak "appropriate bands" and retransformed into competent cells. unfortunately get no colony.