Transformation didnt work - (Aug/21/2008 )

So........ I ligated my insert with my vector ON at 16 C, used 1 µl ligase, 40ng vector coz I didnt have more and 500ng of my insert (some people had told me to use 2µg and other 30ng so I used something in between), then I did a killer digest adding 5 units of enzyme of Bgl 2 (site destroyed upon ligation) for about 40 min.

I added 50 µl of E.Coli DH5 alpha to the mix, left it on ice for 30min, heatschocked for 90s at 42 C, left on ice for 2 min and then added 500 µl of TY medium. Incubated for about an hour and then plated 100 µl of that mix on agar. So far, see no colonies, have 5 inserts and theres like one colony on one of the plates..im waiting till this afternoon

Possible problems:

-I didnt see any difference between the annealed oligos and the ss oligos on a 3% gel, maybe it didnt work? People in the lab said that annealing always worked, I used TE buffer and a thermo block heated to 90 and gradually reduced the temperature to 37.

- I was supposed to digest my vector with Bgl 2 and Xho1. I digested it with BamH1 and Xho1. When I realized I had confused things (didnt see stuffer sequence on gel) I checked and becuase of where the the Bgl 2 site was, it had cut but still left Xho1s recognition site. I assumed when I cut with Xho1 after that the problem was solved. So I cut with Bgl2 and saw my stuffer sequence on the gel.

- A TA in lab told me that sometimes the transformation doesnt work if you do a digest after ligation?

- Maybe 100 µl wasnt enough? Some people in the lab centrifuge their cells resuspend in 200 µl and plate all of that?


Id like to here what you guys think, im quite new to this cloning things. Im not panicking, I thought I would replate the whole trafo, ifthat doesnt work, transform again with undigested vector as a control to see if the trafo worked and if that doesnt work, digest my vector with a Bgl2 Xho1 double digest and anneal again maybe using a cooling protocol with more steps in it, maybe using a PCR cycler. The fact that I didnt see a big difference in the gel might mean more than what people in the lab are telling me? They just said they never checked annealing is simple and it always works?

lol I cant think of anything else at the moment

I have done a digestion after ligation then transformed and I got colonies. Your heat shock time seems awfully long, I use 20 sec for dh5, is there a reason for 90 sec. I do a positive control as standard for my transformations.

You might wish to post this in the molecular biology/cloning forum, you'll probably get more suggestions from there. However, a few ideas:

- Are you sure that your ssDNA oligos are complementary, no simple error while making reverse comp? I think your annealing protocol is ok in terms of temperature/cooling, I didn't use TE. I used this 10x mix instead: 100mM tris-HCl pH8; 10mM EDTA pH8; 1M NaCl (this is a 10x, so dilute it down to 1x).
- Use a positive control for transformation as stevo suggests
- Plate several dilutions of your transformation
- Instead of using just 1 amount of insert, test several, i.e. both 30 ng and 2 ug
- Waiting until this afternoon for colonies to grow will probably only give you satellite colonies without the insert or plasmid