contaminated gel eluted DNA fragment - (Mar/01/2007 )
I have been suffering from cloning problem from last 6 months and I was in search for the real problem which prvents ligation. Finally it seems to me that I found the problem. Yesterday I checked the 260/280 ratio of gel eluted fragment and vector and the ratio were 1.136 and 1.095 respectively. Please experts....is this the real problem with my ligation. If so please advise me how to increase the purity of my gel eluted fragment. I was using QIAGEN standard gel elution kit as well as QIAEXII gel elution protocol. Both are giving low purity. please friends give me some tips...............
I dont really know if it is the purity, but I used Qiaex II kit, always worked fine and then, suddenly, I did not get any clones any more. Other people in our lab had the same problems, so I began to use the PCR reaction direktly as a target, worked fine (but I only had one fragment). This is three years ago now.
We now have changed the kit and did not have any further problems with cloning.
hope that helpes
Bianca
From time to time a have problems with cloning, if I use DNA in a ligation reaction which comes from an agarose gel. So I try to avoid this, by simply ligating the whole PCR-Mix. In a ligation short fragments and (obviously) fragments occuring frequently are preferred by the T4 ligase. So if you are looking for a short fragment or if you've got a fat band on your gel, just try this...
Hmm....
How long is your DNA being exposed to UV? Exposure to UV is very very bad for DNA. You have to work fast and quick. Exposure time is counted in seconds not minutes.
The ratios are pretty bad.
Do you make certain that the wash buffer (must add your own EtOH) is properly removed from the column. Do you remember to spin the column dry.
yes perneseblue, I was taking extreme care to remove EtOH from column and also UV exposure time was as short as possible. Could you plese tell me ddH2O or deionised millipore water is best for elution of fragment from column???? Thank you very much for youe reply 
How many bands does your pcr product have????If only is one I recommend that DON'T use gel purification for ligation.
If what to purify the fragment is better to use a column type purification (as centricon, Qiagen have good kits for pcr purification). Some ligation kits don't need the DNA to be purified so check this first. Other important thing is that each time you purify you loose lots of DNA, so it also could be possible that the ratio vector:DNA isn't as recommend. I would recommend in your case that as I told you in the begining if you have one fragment only make a trial 2 methods 1 w/column purification and the other without purification and then tell me if got results or not. AAHHHH before I forget I your pcr products have more that one band I recommend that use another primer set that gives only one band product. In the majority of contries primers are pretty inexpensive so is better to loose around $50 than 6 month of work without results.
In modern lab settings there is actually little practicle difference between the two.
I would however suggest you use a Tris solution (the elution buffer in the kit is good enough) to elute your DNA. Warming the buffer to 68 celsius before elution also seems to help with recovery rates. And if possible avoid TE. The EDTA in the mix makes the ligation reaction more difficult as it pulls out the Mg ions that T4 ligase needs.
Thank you very much for your useful tips. I will let u know the progress soon...
If what to purify the fragment is better to use a column type purification (as centricon, Qiagen have good kits for pcr purification). Some ligation kits don't need the DNA to be purified so check this first. Other important thing is that each time you purify you loose lots of DNA, so it also could be possible that the ratio vector:DNA isn't as recommend. I would recommend in your case that as I told you in the begining if you have one fragment only make a trial 2 methods 1 w/column purification and the other without purification and then tell me if got results or not. AAHHHH before I forget I your pcr products have more that one band I recommend that use another primer set that gives only one band product. In the majority of contries primers are pretty inexpensive so is better to loose around $50 than 6 month of work without results.
Thank you very much perneseblue, I was not using EB from kit thinking that it contains EDTA.... 
any way thank u very much for this useful information..
In modern lab settings there is actually little practicle difference between the two.
I would however suggest you use a Tris solution (the elution buffer in the kit is good enough) to elute your DNA. Warming the buffer to 68 celsius before elution also seems to help with recovery rates. And if possible avoid TE. The EDTA in the mix makes the ligation reaction more difficult as it pulls out the Mg ions that T4 ligase needs.