No positive clones on blue/white selection - (Oct/28/2005 )
Hi guys!
I met a very puzzling question. I am trying to clone one gene. After ligation, I did a transformation. The number of colonies on the negtive control containing empty vector is 20. But none of them are blue. For the ligation products plates, the numbers are all over 500. These data seem correct ligation has been done. But when I did digestion experiment for checking after miniprep, I could not obtain the correct bands which I expect. I am sure the restriction sites don't have any problem. What is the possible reason for my failure?
Kindest regards!
yang
How did you perform your ligation, and the preparation of the insert and vector prior to ligation?
Did you gel purify both fragments after digestion?
Do the tranformation colonies produce any insert at all on digestion (I know only that the one you're looking for is not there)?
Does your vector encode the lacZ alpha fragment, and does your recipient strain support alpha-complementation?
Is there Xgal in the plates?
Did you gel purify both fragments after digestion?
Do the tranformation colonies produce any insert at all on digestion (I know only that the one you're looking for is not there)?
Does your vector encode the lacZ alpha fragment, and does your recipient strain support alpha-complementation?
Is there Xgal in the plates?
Thanks for replying. I am sorry I didn't describe my question clearly.
I have purified both of insert DNA and vector before lagation. I used two enzyme with sticky end, so I didn't do dephosphorization. When comes to the colonies, some of them gave two bands, neither of which was I want. Some other could not get two bands at all. And my plates contain Xgal, I didn't find any blue colonies. And my vector doesn't contain the lacZ alpha fragment.
Looking forward to hearing from you.
Kindest regards!
Yang
Blue white selection will not work unless the vector (and competent cell) is set up correctly. The blue is the result of beta-galactosidase production by the cell, acting on X-gal in the plate. With the correct vector (e.g. pUC19) the vector constitutively expresses a small fragment of beta-gal which encodes the "alpha complementation factor" from the LacZa gene. This interacts with a chromosomally encoded, much longer, beta-gal gene which is missing the alpha factor, allowing the assembly of active beta-gal. Most cloning strains (e.g. DH5a) have the chromosomal modification necessary for this to function.
The multiple cloning site of many cloning vectors is inside the LacZa gene on the vector. Insertion of DNA into the cloning site disrupts the LacZa expression, eliminating the functional beta-gal activity, and eliminating the blue colored colonies.
Many things can go wrong here. For example, if the insert is in-frame and short enough, active LacZa could still be made, leading to blue colonies with the insert.
But in your case, since the vector has no LacZa gene, you'll wait a very long time to see any blue colonies.
This doesn't explain your digestion results, of course.