cloning from PCR - (Jun/24/2004 )
The biggest problem I see is using 10 µl of the ligation reaction in 100 µl of cells -- try 1 µl of the ligation...
Adding more of the ligation reaction does not equal a better chance of obtaining colonies, in fact, using too much (which you surely are) is inhibitory to transformation efficency.
For PCR product cloning, all should read this article "PCR cloning considerations" from Invitrogen.
Adding more of the ligation reaction does not equal a better chance of obtaining colonies, in fact, using too much (which you surely are) is inhibitory to transformation efficency.
Hm...
Usually, I do the ligation in 20µl volume and use 10µl for transformation, so that, if something wrong happens I can still repeat the experiment. With these 10µl I transform only 50µl Ca-competent DH5a cells. Normally I get about 5 to 30 colonies (or maybe even more) depending on the experiment.
Usually it always works unless there is something wrong with the plasmid preparation.
Is it considered low efficiency?
What would be your additional recommendations about the proportions etc for the ligation?
what seems strange is after digesting, the vector (~3.2kb) shows a clear exised band of about 40bp, the fragment between BglII and XbaI, whereas my digested PCR product shows no small band, even when I ran a 20% acrylamide gel to look for extra small fragments (5bp and 8bp from each primers' 5' end). is this something i should be worried about? how can the enzyme sites NOT be there when I get a very dense band from my PCR reaction at 1.1kb?
You can do the TA cloning of the insert DNA in a TA vector. Then digest the insert with appropriate enzyme to subclone in the cut vector. I think your vector prepration is very good because you cannot see any colony after transformation. I think you insert digestion is not complete because of that is not integrated into the vector because of that you transformation end up with no colonies.
what seems strange is after digesting, the vector (~3.2kb) shows a clear exised band of about 40bp, the fragment between BglII and XbaI, whereas my digested PCR product shows no small band, even when I ran a 20% acrylamide gel to look for extra small fragments (5bp and 8bp from each primers' 5' end). is this something i should be worried about? how can the enzyme sites NOT be there when I get a very dense band from my PCR reaction at 1.1kb?
PCR cloning
I have certainly had my share of cloning issues. It sounds like the procedures in our lab are a little different from those already mentioned, but they work!
First, I usually add only 2-4 nucleotides on the other side of a restriction site introduced into a primer (seems to work fine, but I guess it depends on the enzyme)
When I clone from a PCR product, I either gel purify or phenol-chloroform +/or precipitate it (we never use the PCR clean up kits, but I'm sure they work fine)
I always clean my cut insert/vectors (see above)
I ligate using a 3:1 (ish) ratio... but I have been known to go as high as 8:1 if things aren't working... in a 20ul volume
I ligate O/N and transform 50ul subcloning effic DH5a with 5ul of my ligation and plate 200, 100 and/or 50ul of my transformation (all depends on whether I have lots of plates or have to make them!)
Other than that... just make sure you do positive and negative controls.
I am also a HUGE fan of TA cloning with pGEM. Just make sure you use the right Taq. If I do this, I just throw in my PCR product (no cleaning makes me happy)
As for seeing a 40bp band on an agarose gel... it is totally possible. I can see a 50bp PCR product on a 1.5% TAE gel. It is a little diffuse, but definately there.