In general yes, it's fine. That is standard procedure in many labs. The fine print:

1- You need to leave an overhang (usually 4 or 5 bp long) after the RE sites included in the primers. Most RE will have a tough time cleaving at a recognition site placed flush at the end of a ds-DNA fragment.

2- During cloning, you need to excersize the utmost care in ensuring that expression of your target gene is repressed as much as possible. There have been instances in the literature where basal expression of a non-toxic gene product has been sufficient to select for non-expressing clones (See e.g. http://www.microbialcellfactories.com/content/8/1/8). This is easily done with T7-based systems (most cloning strains do not express T7 RNA pol) but may be tricky with other plasmids.

If your PCR product and the MCS of the vector have been cut at compatible restriction sites (and treated with phosphatase for the vector), you can proceed directly to ligation inside your expression vector.

In general yes, it's fine. That is standard procedure in many labs. The fine print:

1- You need to leave an overhang (usually 4 or 5 bp long) after the RE sites included in the primers. Most RE will have a tough time cleaving at a recognition site placed flush at the end of a ds-DNA fragment.

2- During cloning, you need to excersize the utmost care in ensuring that expression of your target gene is repressed as much as possible. There have been instances in the literature where basal expression of a non-toxic gene product has been sufficient to select for non-expressing clones (See e.g. http://www.microbialcellfactories.com/content/8/1/8). This is easily done with T7-based systems (most cloning strains do not express T7 RNA pol) but may be tricky with other plasmids.

In many years and hundreds of clonings, I have never done an intermediate cloning step unless you are planning to cut out different parts of the sequence for different clonings - at which point doing one PCR amplification, verifying by sequencing and cutting the subclone is more efficient than amplifying all regions individually, cloning individually and sequencing all of them. An expression vector is just a cloning vector with a promoter. I agree with Yann-Vai.

Good luck.

yes, you can...

I use the : IN FUSION from clonetech to to that :

https://www.google.com/search?q=in+fusion+cloning&client=ubuntu&hs=eb8&channel=cs&source=lnms&tbm=isch&sa=X&ei=RYcxU9TzHseLkAfzsYGIAw&ved=0CAgQ_AUoAQ&biw=1024&bih=488#facrc=_&imgrc=da49Pebf19N4nM%253A%3BM67-xLEeufrEjM%3Bhttp%253A%252F%252Fwww.systembio.com%252Fimages%252FCF_process.jpg%3Bhttp%253A%252F%252Fwww.systembio.com%252Fmolecular-tools%252Fcold-fusion-cloning%252Ftechnical-details%3B450%3B481

And it works well!

You can ligate directly the PCR product to the vector. For that, you should add restriction enzyme recognition sites to the ends of the PCR primers (preferably two different restriction sites (for the same enzymes that you use to digest the vactor) in order to have directional cloning). You should also include 2-3 bp to the 5' end of the primers, before the restriction sites, as most enzymes require extra bases to anchor and cut efficiently the product (you can see the number of bases required in URL links of companies selling restriction enzymes).

thanks to all of you

i have added 2-3 bp to the 5'end of primers, before the restriction sites. now i am going to cut directly the purified PCR product, i will elute the digested product from agrose gel after electrophoresis and then proceed for ligation into expression vector.

Dear Sir Yann-Vaï Le Bihan,

if i am not using the alkaline phosphatase in digested vector then about what percentage of vector will recircularize? if i am directly proceeding for ligation withut phosphatase treatment then my experiment will be succesfull or not??

Please suggest me if any other precaution, that i should take during experiments......

I always gel purify the crude PCR reaction in order to avoid any low molecular weight contaminants, which are wont to be cloned preferentially. After that, I use column purification, e.g. Machery-Nagel Gel and PCR Clean-up, (no need to run another gel) after cutting the fragment to generate the cohesive ends. If you use two cloning sites generating non-compatible cohesive ends, there is no need to dephosphorylate the vector. However, if your double digest is not complete, the vector will religate and yield empty clones. To avoid this, after the ligation step, I inactivate the ligase by heating at 65 °C for 10 min and I then treat the ligation mixture with a restriction enzyme whose site in the MCS is supposed to to have been replaced by the insert (and of course, which is absent from the insert).

Yes you can use topo cloning. You can buy a kit and follow the protocol. It is very easy.

Dear Vikas,

It should work also without phosphatase treatment, but perhaps with more clones containing an empty vector after transformation (depending of the overhangs as Pierre said).

So, proceed to the ligation and transformation, and then I would advise you to screen for the presence of the insert in your vector by colony PCR (to avoid sequencing empty vectors). If you got some, choose primers that are in the vector near your cloning sites : this would allow you to know if your insert is present, and if there is only one copy ligated in the vector.

yes, , i use these kits from Thermo with very good results:

http://www.thermoscientificbio.com/molecular-cloning/alicator-ligation-independent-cloning-and-expression-system/

you don't need restrintion enzymes.

Dea vikas:

If you have a unique band in you PCR it is not neccesary to band purifiy it. You could just use a PCR purification kit, It is mu better because the recovery is much more and the protocoll is less time consuming. (consider that if you are amplifying from a plasmid you will still have the template and might get colonies if the selection is in the same antibiotic as your expression vector)

There is also the possibility of performing the restriction directly on the PCR buffer check the site https://www.neb.com/tools-and-resources/usage-guidelines/activity-of-restriction-enzymes-in-a-taq-or-phusion-pcr-mix or just google RE cutting in taq buffer.

After the restriction it is important to get rid of the primers used for PCR so if you perfomed the digestion in PCR buffer here you sould use a PCR purification kit. The other advantage of this is that you won´t need to heat inactivate the Restriction Enzyme.

If the money is a problem you could avoid the PCR purification kit and use a PEG precipitation of the DNA.

always i purify the PCR product using the phenol-chloroform method, and precipitate using the ethanol.

Dear Sir,Fernando Cardoso

i have to digest the PCR product by HindIII and BamHI enzymes (Fermentas), But the PCR reaction mixture i am using from Sigma Aldrich, then i think direct use of restriction enzymes of Fermentas company in Sigma Aldrich PCR buffer may be problamatic, also primers may hinder in digestion. please suggest me in that condition how i should proceed ????

I have the impression that concerns about diminished/altered RE activity when used in buffers of different manufacturers are only lab lore. Mind you, I've never done the actual experiment of comparing enzyme activity by titration in the manufacturer-supplied buffer and that of other manufacturers, but I have quite frequently used mismatched (manufacturer-wise) enzymes/buffers and I am yet to see underdigestion or star activity.

@Martiniano,

Have you ever observed filling-in of the sticky ends left by RE cleavage due to residual polymerase activity when digesting directly in the PCR reaction? I'd love to try that, but I'm concerned that the time saved by the procedure might later be lost having to screen for more clones.

Hello,

You can try with this kit: http://www.clontech.com/US/Products/Cloning_and_Competent_Cells/Cloning_Kits/Cloning_Kits-HD-Liquid?sitex=10020:22372:US

I try several time and I have good result.

@Alejandro

I don´t believe the Taq will be able to polimerize much at 25ºC or 37ºC (usuall RE optimal temperatures) . Anyway If your cloning is sticky your filled ends will ligate with a very low effiency compared to the sticky ones.

The finall result in my case is that it works. At least worked for me the few times I used it.

Hi, the kits http://www.thermoscientificbio.com/molecular-cloning/alicator-ligation-independent-cloning-and-expression-system/

that I used are for: you do a PCR them you cloned directly in Expression vector, no need for restrition enzymes, no phenol.

Clone in E coli JM109 E.coli for sequencing and check the ligation, them Miniprep and cloning in E coli BL21(DES3) for expression.

I do this in 2-3 days,(pcr, cloning) and expression in the 4th day.

with this kit I clone a PCR product directly into an expression vector, without the need for restrition enzymes or phenol.

Dear, Vikas

You can perform your process but verify the  purity of band before to continue. So read this link it could help you:https://www.neb.com/applications/cloning-and-synthetic-biology/pcr-cloning

Hi to everyone.

I have a question: I have amplified the DNA of interest with my designed forward and reverse primers, bringing the restriction enzyme sites I need. My question is: once I cut the amplified product with the two Restriction enzymes, can I use this solution directly for the ligation or I have to run an agarose gel anyway? Consider that I am cutting only my amplified cDNA....I think that in this case I can use directly the solution without purify the cut cDNA as there is not an insert and a vector but only the amplified insert.

Can anyone give me a confirmation of this?

Thank you