hiii

well there is no universal rule that you have to extract DNA from PCR product!! is it?

first thing is your cloning vector, as per my experience pGEM-T easy vector works excellently for cloning of direct PCR products.

Concentration around 50ng/ul is great. But if you want to increase then either try more uls while ligation, or you can increase the total PCR reaction mixture too and then go for gel extraction of DNA..   

I have done the cloning directly from PCR product but size more the 1000 showed a problem.

The one reason is TA cloning is more easy to screen directly.

The second is the PCR product is difficult to cut by all RE enzymes to rule out this we should add 3-4 nucleotides on the 5' end of both primer so that enzyme get enough space to recognize the RE site

And 50 ng/ul is a good concentration if u want more conc than load more sample on gel and isolate collectively.

all the best 

I think that part of the reason is that the efficiency of cloning depends on the size of the product and pcr also produces long reads from the original template dna and some primer dimer both of which may clone using TA or blunt cloning or just bind to one site in sticky end cloning for the longer products generated by pcr. Cloning will work from a dirty mixture but you have more confidence that the right thing has cloned if you clone from a purified product

When you amplify your gene of interest to clone it into a vector, Few things happen:

The raw  pcr product contains admixtures like primer dimers, unused nucleotides, plus fragments of inappropriate range. So you need to gel purify it to exclude admixtures and to select the fragments of appropriate size. Using pcr product directly can wor as well but there is the chance that ligation with unwanted admixtures can occur. In experiments where you do not want this thing to happen you are bound to go through gel elution.

Second You mentioned why elution at every step. The reason is same to validate that you are picking the appropriate sized fragments for cloning. If you are going to sequencing then it is extremely necessary because fragments larger than 1000bp will not be sequenced by the machine, so you need to eliminate them first.

I agree with others that it is a good concentration. If you still need larger concentration you do one thing produce large amount of pcr product e.g. by several separate reactions and load all in one well then go to elution.

Thanks.

The reason that I purify my PCR products for cloning is in general to increase efficiency of the subsequent restriction digest step. As outlined by NEB (https://www.neb.com/tools-and-resources/usage-guidelines/activity-of-restriction-enzymes-in-a-q5-taq-or-phusion-pcr-mix). Briefly, your PCR mix could still contain active polymerase, and combined with remaining dNTPs, could alter (presumably fill in) sticky ends created by the restriction enzymes. Further, presumably the primers used for PCR also contains the restriction site, which could compete with the desired PCR product for enzyme occupancy. Finally, the purification step also helps to concentrate the PCR product a bit (if restriction reaction volume is a concern).

As to your low extraction recovery, I find that kits (especially the columns) don't handle large amounts of material well. Thus, don't try to take too much when you are cutting out your band(s) (counter-intuitive, I know!). Alternatively, cut them into smaller pieces and use more columns. I have also tried running the flow-through through the column again for a second 'binding' step. Also keep in mind that depending on the size of your PCR product, 50ng/uL might be enough material to work with!

All the best!

1) Why don,t we use PCR product directly in cloning, why we prefer to extract DNA in every step?

I am just a novice. I just want share what i have learnt. Please correct if i am wrong:)

We don't use PCR product directly in cloning because somehow we can't be sure if the amplified product is really the GOI (unless you are already established your method). That is why we usually run AGE to see whether the band we get is single or not (your GOI band). If the band is single and is in the correct size, we will cut and proceed to the DNA gel purification. To extract DNA in every step will help to prevent future problems eg: insert the wrong GOI (increase accuracy.)

2)Secondly when ever i extract DNA from gel its concentration is very low, around 50ng/ul and i use Axygen kit. Please give me some suggestion. 

I have used Qiagen DNA gel purification kit. So far, the result was okay. Just we need to properly and accurately follow the procedure. As i learnt when using this kit, the low amount of concentration resulted when there are contamination with the reagent, do not follow/missing the step while extracting. But most of the time, the result for DNA extraction is good.:)

I would prefer using manual method instead of kits to extract DNA with higher yield, and all the process be carried out in cold conditions. The process be extended for three days so that DNA yield be increased at low temperature. 

Qiagen DNA kit is good but results are far better from manual Phenol-chloroform method.

DNA extraction is necessary yes in order to process a confirmed product to be amplified and for further analysis.

Thanks Alot every one . Recently i get very low concentration of DNA ,less than 10ng/ul, and I want to use in ligation so how much DNA i take??

Hi Bushra........

If you want to clone pcr product directly rather than going for extraction  or purification then ensure that there is no any nonspecific band or primer dimer especially when you go for TA cloning. Checking pcr product on gel is best option. If you want to increase concentration of conc. of gel extracted product you can increase no of pcr reactions and elute the product in less amount of elution buffer almost half elution butter recommended in kit protocols. However before going for ligation first calculate how much amount of product you need for ligation that depends on vector size and its concentration as well as size your pcr proudut. Also the ration of insert : vector  is also important. Once you calculate required conc. of pcr product, you can accordingly increase PCR reactions consider the at least 30% loss at the time of gel extraction.

Good luck....

@ sachin , For 20 ul ligation solution i need how much of T4 Ligase, Buffer, Insert (9ng/ul) & vector (2 ng/ul)??how much of this 20 ul ligation solution transformed into how much E. coli cells?

what is the size of insert and vector?

During PCR , all the ingredients may not be completely used up. Some unused Taq polymerase, dNTPs, Primers etc.,  Hence, if you want to get rid of those undesired stuff, you run the PCR product on the agarose gel and see the desired band is obtained. If yield is low, try for a kit of different company for gel elution . Macherey Nagel is good and we use it regularly. Instead of setting up a single 20-25 ul reaction, you can run 3-4 reactions and run on the gel. Excise the desired band and elute in less amount of elution buffer which gives higher yield and can be used for ligation. For ligation, RBC cloning kit works very good. Also, pGEMT -Easy vector is also a good option. Set up the ligation reaction which depends on the size of the vector, insert. You can try for 1:2 or 1:3 or so and check it up which ratio works out fine for you. There are many calculators available online which asks the size of the vector, size of the insert, concentration of the vector and insert you want to use and the ratio you want to use for ligation. Accordingly you can proceed. Here is one of such link 

http://2011.igem.org/Team:UT_Dallas/ligation

Good Luck

Axygen kit is not bad and 50ng/ul   is a good concentration. Do not worry. Proceed next steps. 

this could help