Help : no positive colonies - (Jun/12/2006 )

I'm trying to obtain a positive clone for a long time, and I don't understand why I don't manage. Could you help me please?
I want to insert a 2 kbp insert in a 10kbp vector.
I digest both of them with BamHI in Tango 2X buffer (Fermentas), overnight at 37°C. Then, without purification, I digest with EcoRI in the same buffer (3H 37°C).
I have observed that I lost a lot of plasmid when I purified with Qiagen column. So, I purified my plasmid by phenol:chloroform, and ethanol precipitation. And resuspended it in EB.
For the ligation, I tried a 5:1 vector:insert ratio and a control with only the plasmid (in both case, approximaly 300ng plasmid).
For transformation :
- 5 µl ligation product in 100 µl chimical bacteria
or
- 7.5 µl ligation product in 200 µl electrocompetent bacteria.

I obtained as many colonies for the control (plasmid only) as for plasmid+insert. I didn't tried colonie PCR because before, without control, I didn't had any positive colonie.
Is it my plasmid that is not totaly digested by one of the two enzymes?
After my digestion, I obtained two bands after migration on agarose gel. A big band of the right lenght and another band, less intense at 1000 bp under what we were waiting.
So, I deposed my digested product on an agarose gel and gel purified my two bands (I lost lot of plasmid). After transformation with the big and the little band : no colonies. But no colonies with my plasmid (little or big band)+insert too.

Do you have an idea of what happen? Can you help me?

The buffer seems to be ok. Usually you also need BSA for BamHI. I don`t know if this is included in the 2x yellow buffer. Then, I would do the digestions for only two hours. If you don`t want to purify after the BamHI digestion load a few ul of the digested vector onto a gel to check if it has been linearized.
I personally trust a double digestion with BamHI and EcoRI. Save you a lot of time.
I would purify the double-digested vector and treat it with Antarctic Phosphatase or CIP to make sure it cannot religate with itself. Heat-inactivate AP/CIP and purify vector again.
Also purify the double-digested insert.
Try different insert:vector ratios (3:1, 5:1, maybe 7:1). Use 1 ul of vector in a 20 ul reaction. If you used 1 ul of your vector (=300 ng plasmid) you shouldn`t worry about the concentration. I often used only 30 ng of plasmid and the ligation worked perfecty.
I don`t know why you see two bands on your gel. How far are the BAmHI and the EcoRI restriction site away from each other? Maybe the vector is still partly in the supercoiled form (undigested)?
Try to get a clear band after digestion. Makes no sense to ligate and transform fragments of DNA of which you don`t know what it actually is, rigth?

looks like a straight forward ligation to me. if you are loosing digested plasmid during purification, the only solution is digest more and purify it. I generally don't carry out gel purification instead I add 3vol of QG to digest mix and 1 volume of isopropanol and run it through qiagen column and elute with Tris/ elution buffer.
Regarding the ligation, both are very good enzymes and there incomplete digestion is not a problem. I use quick ligation kit from Roche and it works like charm (I used other kits from Promega, NEB - this works very good). I generally run the vector and insert on a gel next to each other and then determine the ratio to use and then go for ligation ( your vector is 5times insert so intesities vary accordingly). I would suggest changing the ligase and the buffer (use fresh buffer and try to handle it carefully as many of them have ATP in them) and also running the vector and insert next to each other.

I would like to know what the second band is. Do you also see it if you load undigested plasmid?
For the transformation I use 100 ng (vector + insert in ligation mix) and 100 ul chemocompetent cells.
Your cells are propably compentent because you see colonies.
Where do you get your insert from? Do you cut it out of a vector or is it a PCR product?

Can you show us a photo? Maybe there is a contamination in the restriction enzymes?
How many extra bp did you add in front of your restriction sites in the PCR product?

prepare new C cells and change new bacterium culture medium.
I have problems similar to you before, I obtain too much clones on plate , but PCR checking show no positive clones! . later I found the culture medium has been contaninated!

I tried to digest with BamHI of our lab and BamHI of another lab : it gaves the same result (two bands).
I added 3 extra bp in front of the restriction sites in the PCR product.
I don't thing the problem comes from the cels because another person obtained good results with the same cells and I have tried with electro and chimical competent cells.
I have already changed my culture media.

Other ideas?

i would def go for more insert and less vector - 300ng seems like a huge amount of vector to me - remembering that kits such as pgemT recommend only 50ng of vector max......
also, try the other guys suggestions of double digest and CIP treatment - should be straightforward...

also, with your concern regarding two bands etc - is your plasmid actually what you think it is? sequenced? right size?

good luck