You have to extract and purify the band of interest, which you have cut from the gel. I believe there is some contamination from the gel. You should try it again.... amplify your product and run on the gel, make sure to just only cut around the band of interest and use fresh reagents (Qiagen purification kit).

Good Luck

I do cloning after PCR. But I do a DPn1 digest after PCR and use the Dpn Digested mix to clone in vector.

Omid: "You have to extract and purify the band of interest, which you have cut from the gel. I believe there is some contamination from the gel."

This is what I done

Omid:"I believe there is some contamination from the gel. You should try it again....

This is the second time. "

"make sure to just only cut around the band"

Yes I cut the minimum of agarose amount (100mg as Qiagen preconised)

Qiagen say to me that it is salt! Add one step of centrifugation to overcome this problem!

I'll try and keep you inform!

High absorbance at 230 is common after gel elution. Still the product works well for TA cloning.

I wouldn't worry too much initially about the 230 nm peak contaminant, unless it interferes with your cloning. If it does, you can always purify your sample further using many of the DNA purification kits from Qiagen, Zymo, etc.

What is important is that after cloning, you ensure by restriction digests and sequencing that the insert is indeed what you PCR-ed up.

Good luck!

Try to use a new kit (QIAquick Gel Extraction Kit)?

Try to re-purify your sample by washing and passing through the column using new buffers. Phenolic & Guanidine HCL in the solution will absorb at about 230 nm.

Here is a document that showed how salt may absorb at 230nm.

If you have a well separated, strong, PCR band, you could also try in-gel ligation. It may save the worry about purification. Just need to make sure that you are good at estimating the relative molar concentrations of the vector/insert, and keep time on the UV box to minimum.

Thanks for all your comments.

For now, My PCR runs. I'll plan to make one step more of centrifugation at the end of the purification protocol, to ensure that salt removed.

result in about 3h!

Hi,

Wish you luck.

I will not be worried so much as long as the desired fragment is ligated successfully

i don't think you have to worry much about it, you can proceed with cloning. Have you checked it in agarose gel after purification? what is the value of A260/280 ?

A260/280 is correct! around 2.00!

It has to be some kind of salt precipitation, which is not supposed to be in high concentration from a gel extraction protocol. You might consider doing and extra wash or two when the DNA is bound to the column and make sure that the column is both dry and in a clean tube before eluting the DNA. The DNA should then ligate well, even if you have a bit of absorbance at 230. An extra ethanol precipitation will help, but I don't think this should be necessary for simple cloning.

It is not unusual to observe a 230nm absorbance peak following gel extraction of DNA using the Qiagen kit. Whilst the composition of QC buffer is proprietary, it probably contains guanidine HCL, a denaturing agent commonly used for DNA purification that has a very strong absorbance peak at 230nm. In my experience, it has no noticeable effect on downstream cloning. Best of luck.

Adding one step of washing using PE buffer just before the elution buffer doesn't work.... I have aloways my 230nm pick absorbance.

So let's go for the cloning with it!

I see similar peaks as well all the time. It does work. I always correlate what I measure on the Nanodrop with a similar aliquot run on a gel.

I find that if the 260/230 is much less than one the ligation does not work. I think that the ratio gets worse the longer you heat the band to dissolve it. Chop up the band and melt only a small volume of gel (200 ug) per tube; it will probably take 2-3 minutes instead of 5.

Also, I prefer the Zymo Research Zymoclean™ Gel DNA Recovery Kit. It uses sodium iodide instead of guanidine to dissolve the gel, and can be done at a lower temp. You can also recover the band in 6 ul. For older researchers, you may remember when NaI was used with "glass milk" for this purpose. Now it is in column form.

Have you checked you purifying product by running on gel? Mostly there might be the problem of poor DNA yield. If it is singed band that use NA-acetate pptn.

Emmanuel,

I suggest that after PCR load half (or less) of your PCR products on the gel. If you get a single sharp band on the gel, clean the PCR products that you held back (lots of kits available) and then use that DNA for cloning. The DNA purity will be much higher than you will get from purifying DNA from agarose and will thus greatly improve the efficiency of subsequent cloning. Pick several transformants and perform ‘colony PCR’ to determine the size of the insert within the plasmid and verify that the size matches the band that you want. Even if you are cutting out one PCR band from three, the higher efficiency of cloning after ‘PCR cleanup’ of DNA may make it worth your while to use the gels simply to see what you’ve got and use cleaned up PCR products for cloning. Similarly, the co-migration of different sizes of DNA fragments can mean the even within a single excised band there may be multiple sizes of molecule. The products of ‘colony PCR’ can also be subjected to Restriction Enzyme analysis so if you know something of your target DNA fragment (and even if you don’t) you can quickly determine how many classes of DNA molecule you have cloned.

I have cloned a 1.3Kb fragment of gel eluted DNA. I did the elution using Qiaquick and it works. Just try cloning it. You can worry about the quality once you find that cloning is not working.

Don't worry about this. You can clone your DNA without problem. I have always used this kit to purify the DNA and then I have cloned it without problems. Good luck!

This is not unusual. You can run a small volume of your purified PCR product on agarose gel and check if you have the right size band. You can proceed to the ligation step.

T4 ligase doesn't care much about the quality of your DNA. Just through it in the mix and transform.

Thanks for all your response.

After 3 weeks of cloning and sequencing, I can say that it worked, and that the A230 pic absorbance was not a problem.

Using Promega kit for cloning I have very well efficiency of ligation since I had a minimum of 30 "white colonies" per sample.

Again, thanks for your help!

Emmanuel- Two options if you must purify your band from a gel: [1] Run it through a low-melting agarose in TAE, cut out your band, melt it at 65C, and digest the agarose with beta-agarase (NEB). You can then extract once with phenol:CHCl3 and centrifuge, then preciptate with NH4acetate and EtOH. [2] Electroelute your DNA from the gel plug (look at an old copy of Maniatis) and precipitate. The latter is the cleanest. Both approaches are old school and more trouble, but give very clean DNA, and often the final yield is better. We typically use the modern gel extraction kits, too, for convenience, but for anything giving us trouble or unusually important we go old school. Just some options to consider.

The wash steps in the gel extraction steps will help with this issue. Do the extra wash which is listed as "optional". The absorbance at 230 nm doesn't harm the ligation in our experience, and is likely guanidine. All the extra precipitation steps or switching to special agarose probably is not necessary for your simple cloning approach. Don't make it more complicated than it needs to be - you just need a successful ligation so that you can get this back into bacteria for amplification and sequencing.

Yes, we did. We cloned a DNA fragment of about 1.4 Kb eluted from agarose gel. Everything worked well.

I really wouldn't worry, as long as you have enough for a ligation reaction you should be golden, However, if your elution volume is ~40-50 uL, the that its yield is pretty low... I usually gain yields ranging from 150-300 ng/uL, but then, I normally elute with about 40-50 uL ddw water :/...but the peak at 230 nm is something you always get after gel extraction, as Daniel mentioned, it's probably Gdn-HCl, and not at a very high concentration (not enough to interfere with most downstream applications at any rate)

Hi there. I think there is some great feedback here and I am sure you have already work it out. I have had this often when using the Qiagen kit for purification, as mentioned above even though the components of the QC buffer are proprietary, it probably contains guanidine HCL which absorbs at 230nm, I also wonder if it has to do with the pH indicator added to the QC buffer. I have managed to get better purity by either washing the column twice before elution or even (although slightly more difficult) when I have a sample that I want really pure I will not add the pH indicator to the QC buffer, but rather dissolve the gel piece in clear QC buffer, then remove a small volume of this solution and add the pH indicator to that. This will tell you if the pH of your solution is fine to continue on with or if you have to lower the pH before continuing without adding the pH indicator to the entire purification.

Aside from that, provided you have some DNA in your purified sample then this particular ratio does not seam to affect the cloning efficiency from my experience.

Best of luck.

To elude DNA with warm buffer/water would help your quality peak at 230 will be lower till none. in your case to clone , this 230 peak will not be a problem coz the cloning process is very specific , and it's in a big dilution already so it will dilute this effect , so your exp should not have any problem , good luck :)

Thanks for all the comments. I had the same problem today. I was not sure whether to carry on or trash the samples and start over. After seeing that many people saw similar low values, I decided to carry on. We will see how it works tomorrow...