Cloning PUC19 experiement, can you help me troubleshoot what went wrong in the blue/white screening.?
I ran a blue/white screening experiment with PUC19. I ran two controls to test the E. Coli cells were competent. One in -AMP and one +AMP. a bacterial lawn was present in +AMP as expected.
I then ran an UNCUT PUC19 plate These show as blue colonies as expected.
The next plate was BAMH1 cut PUC19. showing less blue colonies than the uncut PUC19. But no white? Does this mean the cells contain the plasmid, but not the insert since i only grew blue colonies?
I then did a self-ligated PUC19 plate, also containing blue colonies, this is also to be expected?
Then plate 6 contains the cloning experiement itself. But there are no white colonies at all on the plate. This is not to be expected? Could my ligase be defective?
(X-GAL and IPTG was used)
Any help would be appreciated in explaining what's happened.
- Ted KLv 71 month ago
Yes, the ligation may not have worked, but there are other possible issues. First you can check your supposedly recombinant plasmid on a gel to see if it's the predicted size for a successful ligation--obviously it should run slower than bare plasmid. As a further check in another lane, run that same recombinant plasmid after the appropriate restriction digest--you should get two fragments--one fast-running one for the insert and a slower (larger) one for cut PUc. if that shows that what you're transforming your bugs with isn't what you thought it was, then you can try another batch of ligase. But what about your buffer for the ligation reaction? Was it fresh? Over time, the ATP and DTT in the buffer can degrade. What molar ratio of insert/vector did you use? Depending on the size of the insert, I think most protocols recommend a ratio somewhere between 1:1 up to 10:1 (insert:vector). Finally, if you isolated your recombinant plasmid by cutting a band from a gel, did you do it as fast as possible? If you had the gel on the light box for too long, the UV light can damage the DNA. That's all i can think of right now, good luck.