no colonies, ever! - (Feb/27/2014 )

Hi everyone, I just started working in a lab for the first time (so please forgive my poor technical language and gaps in knowledge!!), and am having a lot of trouble doing a simple transformation. Each time I've ended up with little to no colonies, with the existing colonies being false-positive. My procedure goes something like this:

Vector: pEGFP-N3 

Insert: ERCC1 (v1/2/3)

1. PCR ERCC1 (40ul of reaction, used this much because I have trouble with low yield in the following purification procedure)
2. Gel purification 
3. Double digest (SalI, BamHI) vector and insert
4. Gel purification of pEGFP and column purification of ERCC1
5. Ligation (total volume 35ul, used 4ul in transformation)
   • 0.2ug of ERCC1 variants (~30ul)
   • 0.03ug of vector (1ul) 
   • T4 ligase (new)
6. Transformation (heat shock method)

I know the competent cells work because I ordered it from a manufacturer, and transformation of their control gives me a lawn. Transformation of parent vector also gives me a lawn.

I also began to doubt whether is my own technical issue, and asked a grad student (who's been doing it successfully) to help me transform my ligation mix. And -> no colonies.

So I'm wondering what is up with my ligation mix? I even measured the concentration of DNA using nanodrop and followed the vector:insert ratio according to the 1:3 rule.Can it be possible that I've damaged my DNA from UV exposure? However, each time I ran a gel to check the presence of DNA I get a clear band (definitely some loss there, but the bands are present).

At my wits end! Someone help a newbie out :(

SalI is a known problem enzyme for cutting PCR products near  the ends. Do you have another choice? How many extra bases 5' of the SalI site are there on your primers?

Thanks for the reply! I designed my primers with SalI in mind so I would have to make new primers.. and I added 3 extra bases.

I would also recommend - if your PCR is a single clean band, just doing a column purification, then digesting.  If you have the parent plasmid (i.e. the one you are PCRing from) coming though, do a Dpn1 digest to cut up the plasmid before the purification.

Ensure that the cut sites are on the 5' end of the primers for both primers!  It's an easy mistake to make.

For the ligation, work out the molar ratios:  ng insert = molar ratio x x vector ng.  Where molar ratio is a number, usually 3 or 10 and work from there.  Typically for ligations smaller amounts work better, aim for less than 20 ng of vector.

hi everyone, thanks for the replies. still having no luck with this transformation. have tried everything (varying the molar ratios, minimizing UV exposure). plates as clean as when i first poured them. 

It could be that the insert is toxic and your bacteria are dying.  Are you sure that you are using Kanamycin for the selection?