Troubleshooting Ligation/Transformation - (Aug/30/2006 )

Hello,

I am having trouble making what should be a very simple construct and I believe the problem lies with the way I am doing ligations but I have been unable to figure out what is going wrong. I have tried more than a dozen times now over two months and have not gotten a single colony.

I am trying to replace an insert in a pBAD24 vector (pBAD is about 4500bp while the current insert is 500bp if that matters) with a new insert of about 300bp. After sequential digests with AvrII and then SalI, I run the DNA on the gel, cut out the digested pieces and perform a gel extraction using Qiagen's Minelute Kit, eluting using the elution buffer provided. As far as I can tell, the REs are working properly. I also know I have recovered the DNA from the gel pieces (I always run some out on an agarose gel).

I then ligate using NEB T4 ligase and leave it overnight at 16C. I have tested the ligase and know it works. I usually use about 15-20 ng of vector and have tried vector:insert ratios from 1:3 to 1:10. I then use 1 ul of the ligation reaction for transformation and let the cells recover at 37C for an hour. I use electrocompetent DH5 cells made in the lab. I have been told have a pretty good transformation efficiency and my informal testing confirms this.

Invariably, the result is the same: I get no colonies the next day.

Can anyone see if there is something off or suggest things I could do to pinpoint the problem? Thanks in advance. This is driving me crazy.

Your protocol sounds quite ok... Can you try and electroporate with 2ul? Or maybe you could use more material to ligate

I always do a PCR purification after digestion -- even if I am doing a gel extraction -- before I do my ligation. After ligation, I would also do another digest with a single cut enzyme that will cut your vector (not your insert), and run on a gel to verify the size. Run it next to vector alone (cut with the same single cut enzyme) and you should see the 500bp difference to make sure your insert is ligated properly. Then you can proceed with the transformation.

Hope that helps!

Hmm, I've had good luck checking the ligated DNA on a gel before transformation, but your concentrations needs to be high, which could be the problem?

I actually use a 1:1 ratio of vector to insert, but I think that works because of what chick gene said about the sizes being so different. It took me a while to optimize the ligation, but for what it's worth, this has worked well for me with pET14B as the vector:

12uL digested (and purified) vector
12uL digested (and purified) insert
1uL New England Biolabs T4 DNA Ligase
3uL 10x T4 buffer
2uL water

I "incubate" it at room temp. for 30 minutes, and then run a small amount on a gel. The band is usually a bit faint (because I run so little, and it is diluted due to the ligation mixture).

I would suggest using a total of 100 ng DNA in a reaction volume of 10 ul
When you calculate the amount of insert to use in ng, doyou do it this way considering the ratio?

100 ng * (500/4000) * ratio insert/vector (3/1)
, giving you ng of insert to be added

for transformation 15 min on ice, 1 min 42 C and 3 min ice
I recover cells for 2 hours

Check for damaged ends from the restriction digests and/or UV damage during gel purification by preparing single-cut vector and transforming plus and minus ligase.

Thanks for all your suggestions.

chick gene, I am not quite sure what you mean about the vector being more concentrated due to its size. Do you mean in terms of mass? I usually calculate the ratios in terms of molar concentration.

tertu, I normally decide how much insert to add by basing it on the amount of vector. Your method sounds like it might give me more reproducible results (assuming I get things to work).

I'll update when I get a chance to retry the ligation. Thank you all again.

I guess I am here to spread a horror story and (perhaps) an urban myth.

After several unpleasent experiences I now avoid using SalI like a plague. SalI works with PCR products (ie SalI digested PCR product ligate well) but I have never gotten plasmid DNA that has been digested by SalI to ligate efficiency.

I did use the special SalI buffer and all but with little effect.

So is my bad experience just me or is it SalI. If you have done everything right, I would blame SalI. You could try using a different enzyme if or use partial fill in to make the ends compatible.