From your second post I think what you do is, after double digestion you are purifying your digest via a gel extraction. the reason behind your not getting a band after digestion may be incorporation of inadequate amount of DNA in the digestion mixture. If you are not adding a considerable amount of DNA for digestion and then gel extracting the digest, chances of loss of DNA during gel purification is quite high and that may be the reason why you are not getting a band in the final gel and self ligating vectors after ligation. I would recommend that you quantitate the DNA concentration after you gel purify the PCR product (you can use a PCR purification Kit to eliminate the proteins and buffers; also minimizes DNA loss; also can concentrate the purified PCR product bu performing the extraction step in a lesser volume of buffer)and set up your assay with maximum amount of DNA for an overnight digestion (8 hrs). After Digestion run the gel and after gel purification check DNA concentration again before setting up the ligation reaction.

i run about60micro pcr product,and clean from gel with gel purification kit,i get good band,then i do double diget with Nde1 and Hind3.after digestion clean with gel purification kit so.although now don't get band, keep on cloning and another steps that said

I guess one of your enzymes is not cutting. When you say you take 30 colonies and all of them have empty vector, that means plasmid is self-ligating because it has been digested in only one site. I assume you check on agarose gel that your expression plasmid is linearized. I recommend you to clone first your PCR product into any vector (I use pGEM-T) and then do the digestion with both enzymes. In this way, the insert obtained after digestion will have both restriction ends. For your expression vector, try to insert any DNA fragment in one restriction site and then digest with both enzymes. Again, this will tell you if vector has been cut at both ends. Then, when you are sure both insert and expression vector are doubled-digested, try the ligation.

Ok, a few questions: How do you add the restriction enzyme recognition sites to your PCR product? By additions to the 5' end of your primers? Did you add some additional bases next to the site (some enzymes need this to be able to cut DNA).

Did you check in single digests with your vector that both enzymes are working and linearizing the vector on its own?

Did you try to use alkaline phosphatase to prevent religation of the vector?

How do you do the clean-up?

It's very important to run controls when cloning using restriction enzymes. You could have 1) uncut vector transforming into your E.coli or 2) vector self-ligating (ie: no insert) after digestion and then transforming into your vector. Using alkaline phosphatase is highly recommended to prevent 2) (cloning will be difficult if you don't use it), but you need to check that this step has been effective and 1) still needs to be ruled out. Just because your vector looks completely cut on a gel doesn't mean restriction has been 100% effective and any uncut vector will transform in very easily.

When you come to do your transformation reactions, perform one transformation using a "no ligase" control. (ie: Try to transform from a ligation reaction that has had no ligase added). If your vector has not been cut efficiently, you will get colonies on your plate with this control. Also run a "no insert" control (ie: a ligation reaction with no insert added). This will control for cut plasmid re-ligating to itself. If you get colonies on your plate with this control then your alkaline phosphatase treatment has not worked efficiently.

Dear Parastou, first clone the purified PCR prduct into cloning vector (like pGEMT or any other) then transform this recombinant vector in competant E coli cells (having no recombinases), screen the transformed colonies from E.coli. isolate the plasmid with your gene construct. then digest it with restriction enzymes that you have added at terminals of primers. purify the digested product from agarose gel (by doing so you can get gene with enzyme overhangs), simultaneousey digest the expression vector in another tube using the same restriction enzymes, purifiy it also from agarose gel.

Finally add the purified gene overhangs and expression vector in 3:1 using T4 DNA ligase. transform it again in E.coli and your target oraganism. All the best

From your second post I think what you do is, after double digestion you are purifying your digest via a gel extraction. the reason behind your not getting a band after digestion may be incorporation of inadequate amount of DNA in the digestion mixture. If you are not adding a considerable amount of DNA for digestion and then gel extracting the digest, chances of loss of DNA during gel purification is quite high and that may be the reason why you are not getting a band in the final gel and self ligating vectors after ligation. I would recommend that you quantitate the DNA concentration after you gel purify the PCR product (you can use a PCR purification Kit to eliminate the proteins and buffers; also minimizes DNA loss; also can concentrate the purified PCR product bu performing the extraction step in a lesser volume of buffer)and set up your assay with maximum amount of DNA for an overnight digestion (8 hrs). After Digestion run the gel and after gel purification check DNA concentration again before setting up the ligation reaction.

@Shakri: There is nothing bad about a gel elution. If this is done properly, you have a recovery of 90% of your DNA. And this works also with very small amounts.

@Christian, Thank you. I did not mean to say that Gel elution is bad. I always prefer a gel elution after digestion. But like you said, if not handled properly, there are chances that you might lose a portion of our DNA. That's why I recommended the use of a PCR Purification Kit.

If you're not getting a band on the gel, you need to address that before moving on to further steps. I think Shakri has a good point-- you should check to make sure you haven't diluted your gene too much after the gel. If you cut out to much of the gel outside of the band, you will dilute the gene, potentially to the point where you need to run the digesting multiple lanes where a band won't be visible. So make sure to cut as narrow a band as possible. If the gene is dilute, you can use the purification kit like Shakri suggested or you can precipitate the gene from EtOH and redissolve it in a smaller volume.

When you say that you don't get a band, is the lane empty or is it smeared with no resolved band? If it's smeared you may be getting star activity. That could explain the fact that you get some colonies from the ligation if there are continuous smaller fragments present.

Try to increase the initial DNA concentration as there's two steps of gel purification. Try PCR clean up kits, which give a much better yield. Check the PCR product on gel before the ligation. Include a no ligase or vector control as Cheryl has explained.

What template do you use for PCR? Is it cDNA or another construct? If using plasmid template treat your sample with Dpn1 to get rid of the remaining backbone then PCR purify then run just a small amount to make sure you have the right band not all your PCR reaction (you don't have to run all the amount then gel purify it, PCR purification is more reliable and will save your DNA). Digest both, gel purify your new backbone and run a small amount of insert then proceed to ligation. My assumption, the colonies are self ligation of the backbone. So you may need to check if your restriction enzymes aren't compatible after digestion otherwise you will have to treat them with phosphatases. Also, don't use these new nuclear stains to visualise your gel like Safe view as they interact with DNA and damage sticky ends. Don't expose your DNA much to UV light as it damages DNA. Don't rely on nanodrop to determine your DNA concentration. Try different ratios for ligation reaction but 1:3 should be the optimum.

Hope this helps and good luck :)

Parastou does not mention gel purification at all. If this is a correct description then the DNA is degraded during restriction after PCR. Use DNase free buffers and water.

Shakri & Christian are right that in good labs with good procedures gel purification works fine. In standard PCR cloning strategies gel purification is superfluous and the time and trouble can be saved. The stuffer fragment and the end fragments of the PCR product are small and easily lost in any purification, being a PCR column purification or an simple ethanol precipitation. Cheryl's recommendations of gel controls for the + / - ligation reaction and transformation controls for + / - ligation saves a lot of time in lab not wasting time on checking empty plasmids! Another useful control is +vector -insert +ligation; this should yield no sign of ligation on gel and only a few more transformants than the

-ligation reaction.

For a standard two-enzyme cloning P'ase can be saved; if trouble better check that BOTH enzymes actually cut's efficiently!

A final word on gel purification: IF you really wish to do this make sure that you are using a ~360 nm UV-lamp, any short wave-length like ~260 or ~312 nm lamps kills you DNA and burn your hands rapidly. Clean buffers and clean equipment also helps.

Sorry I didn't see the second post by Parastou, anyway anything but the DNase free buffers is still valid, Cheryl had also the good controls.

Purify your PCR product, and then insert in to a vector so that later u can transform them into E.coli so u will have a successful transfer....

You have got a good band after PCR. However, you did not confirm any band after the digestion with restriction enzymes.

1. Check the insert DNA sequence if restriction enzyme recognition sites is contained in the PCR product.

2. Seach a step where the band disappeard.

I think that a purification step is problem.