another better option is first clone it in any T-tail vector and go for sequencing. Only after analyzing the sequence you will know whether it will produce a protein and if so what will be the size. Later, design primers with tagged restriction enzymes of interest so that your sequence to be cloned is "in frame" for complete protein synthesis. This will be a better pathway than first trying to express them in expression vectors before sequencing. rather, it will cost you much the other way.

Having no blunt end restriction sites is advantageous because sticky ends ensure proper orientation of your insert during the ligation process. So you can pick two sites whose enzymes are available at your lab. Look to it that their recognition motifs are not being methylated. Then you design primers against your gene with corresponding restriction sites. You digest and dephosphorylate the pET vector and you just digest your amplified PCR product. Clean both by gel electrophoresis or PCR clean up kits and ligate.

In case your insert has blunt ends you could cut the pET vector smoewhere in the MCS and polish the resulting sticky ends by treatment with T4 DNA polymerase before ligating vector and insert (but this is a quick & dirty procedure and you would have to check your construct thoroughly).

Hope this helps.

Philipp

Hi, Kashif!

In addition to Philipp suggestions you can clone your PCR fragment into pGEM-T (or similar vector), then compare MCS of your vector and pGEM-T and choose restrictases you have in stock or purchase new, all depends on you:)

Good luck!

The problem is I wish to clone the PCR product and PCR product is always Blunt ended, I dont wish to use any restriction enzyme against my PCR amplified product because it might cut my product of interest or I can say amplified product in between the gene of interest and the primer I have used to amplify the product of interest don't have any restriction site means any sticky end restriction digestion sites.....

If you need to make construct with gene coding your protein of interest, just incorporate in 5' oligo site of Nde1 or Nco1 (their sites contain ATG), so you will have no unnecessary additions to your gene.

Or choose another available vector with sites for blunt-end restrictase in MCS.

Is the sequence of your gene unknown or why do you want to avoid any restriction of the PCR product? Because I don't see any differences in ligating the blunt ended PCR product in a pET vector you have to make blunt ended in the first place and just attaching appropriate restriction sites immediately before and after your gene. In the end you have your gene in the MCS surrounded by other restriction sites. By using your own sites you could reuse them for further cloning.

Respected Philipp, Sequence to my gene is unknow....since i have isolated from sediment sample and i wish to see whether it get express properly of not then only i wish to go for sequencing.

pET expression vectors have no blunt end restriction digestion site? What do you mean? The vectors are T-vector or these vector have no appropriate blunt end sites for your gene?

(1):If these vectors are T-vector, you don't worry the gene-fragment's ligation. Because normal PCR will add an A base behind the 3'-terminal automatically. So your PCR product will bind to vector with T4 DNA ligase.

(2): If you can't get the proper restriction site for your gene and you unwilling to cut your gene, you can add two restriction sites at the both side of your gene by PCR. Just add an extra sequence to your primers' end, and then process PCR. And finally you will get a gene with the special restriction site (with both ends) suited for the special vector.

Got it. Try the polishing step of your pET vector with T4 DNA polymerase. I did this once in order to eliminate a gene coding for a toxic protein (preventing "empty" plasmid amplification ;-) ) and it worked well.

Dear Ding,

pET vectors generally don't have blunt end restriction site at MCS region of the vector.

I consulted my pillow and you don't have to clone your gene necessarily. If I got this right, you took your sediment sample, added primers and maybe got multiple PCR products. Now you want to focus on one signal, right? Hence you could just elute and pruify the PCR product and send it for sequencing with your primers.

Moreover you could do cell free expression in combination with a linear template kit. You would perform a second PCR with primers of such a kit that add regulatory sequences to your gene (promotor, RBS, purification tag and so forth). Of course you would have to redesign your primer to the effect that the first PCR product (your gene) can be recognized by the primers of the kit. The second PCR product would serve as template for transcription and translation, optimally perfomred in separted recations, but a simple batch setup would be sufficient for a brief glimpse of expression. It is expensive but cool ;-) and you could perfom this for all your first round PCR products in parallel ...

there will be three type of vectors in any expression vectors to make your sequence in frame. So, accordingly choose the restriction enzymes and sequences of those respective enzymes should be tagged with the primers and amplify again your pcr product. After which, digest both i.e., PCR products and the vector. Gel elute or purify them, and followed by ligation and transformation.

Gud luck

another better option is first clone it in any T-tail vector and go for sequencing. Only after analyzing the sequence you will know whether it will produce a protein and if so what will be the size. Later, design primers with tagged restriction enzymes of interest so that your sequence to be cloned is "in frame" for complete protein synthesis. This will be a better pathway than first trying to express them in expression vectors before sequencing. rather, it will cost you much the other way.

Hi Kashif,

I think there is no problem in using restriction enzymes on your PCR products. after amplifying your gene of interst you can go for TOPO-TA cloning which is efficient due to its TA ligation. But remember to use Taq polymerase in your PCR to get get an 'A' overhang in your PCR product. Further you can go for sequencing to check the orientation of the gene in the TOPO vector and then subclone in your end vector.

Cheers.

I agree with Rangan and Philipp : it is much better to know the sequence, which at some point you will need anyhow. For this, T-A cloning is a valuable option. Since Taq has only poor fidelity, I would advise to amplify the fragment first with a high-fidelity enzyme such a Phusion, and then add the A extension by incubating the fragment with Taq, as advised in the instructions of the T-A cloning kit provided by Invitrogen (now part of life Technologies).

Once you know the sequence, you can design your primers with restriction sites for cloning into your expression (pET) vector while avoiding sites present in your gene. The most convenient sites at the 5' end are NcoI and NdeI, which comprise an ATG codon and avoid adding extra codons to your protein. If your gene contains one or more of these sites, you can polish the vector site with T4 polymerase and perform blunt ligation with the 5' end of the gene, which you will have amplified with a high-fidelity enzyme (not Taq) that yieds a blunt end and a 5' primer that starts at position 2 (for filled-in NdeI) or 4 (for NcoI) of your ORF.

As to the 3' site, I prefer to choose the most distal site of the vector MCS (usually XhoI in pET vectors), so that after performing the ligation, I can heat-inactivate the ligase and treat the ligation mixture with a restriction enzyme whose site was originally present in the MCS, but absent in the insert. This gives a very low background of empty clones

Use NEB's Gibson Cloning kit (E5510S) or similar technology and you don't need any restriction digestion and you can clone it into any vector.

We have had good success with GeneArt seamless cloning.

http://products.invitrogen.com/ivgn/product/A13288?ICID=search-product

If you want to go with conventional cloning strategy, include appropriate restriction sites in your primers. In case of proof-reading polymerase, you can go for A-tailing if you want to clone in an intermediate vector (TA vector). After sequencing, you can digest the intermediate construct and pET vector and go for ligation and transformation. Otherwise, you can cut the PCR product with restriction enzymes and clone it into pET vector (my success rate is less in this strategy).

Good luck!

Just to begin, exactly what pET vector (pET100, pET200, pET101, pET102, pET151) are you using? I hope the answer is one of these Invitrogen vectors.

For example, I use pET100, an IPTG-inducible expression vector with a native start codon followed by HigTag, enterokinase recognition site and Xpress epitope, all of these are downstream of the native start codon. THERE IS NO MULTIPLE CLONING SITE on this vector, and there is no need to use ligase in the cloning reaction. This vector has a GTGG overhang on the complementary strand. What I do, according to the instruction manual, is to amplify the gene of interest with a CACC sequence before the first codon. CACC is complementary to GTGG, and a topoisomerase will bind these sequences together. The CACC sequence must be added before the first codon of your forward primer (or reverse, if the gene you want to express is coded in the complementary strand of the source DNA). Therefore, the sequence of the primer always should begin with CACCATG, all primers I have checked in reports with this same type of vector include the start codon of gene to express (I might think there is no need to have this start codon, since expression is being started by the ATG of the vector, however it would be interesting to check if removing it makes the protein still expressible and active).

Another thing to have in mind is not to have a CACC sequence on the reverse codon. Quoting Invitrogen manual, "To ensure that your PCR product clones directionally with high efficiency, the reverse PCR primer MUST NOT be complementary to the overhang sequence GTGG at the 5′ end. A one base pair mismatch can reduce the directional cloning efficiency from 90% to 75%, and may increase the chances of your ORF cloning in the opposite orientation."

I must say there are so many approaches to achieve the goal. First, what you want to do with the recombinant plasmid? Do you want to express the protein? I suppose so because you are saying you want to express it in pET vectors. So you must first amplify the gene with correct restriction sites which are in frame with the vector. If you have not amplified the product with RE sites in mind, then it is a good idea to use either TA overhang vectors (many in the market) or simply use the pGEM vectors supplied by Promega. In this vector, suppose if your insert does not have a SmaI site, they you can keep your insert, vector SmaI under ligation conditions together. This approach is blindly successful even for novice ( Forgive me, I am not underestimating your knowledge) because, every time the insert gets ligated to the vector the SmaI cannot cleave the recombinant plasmid. So the reaction drives to left and after transformation, you can have many white colonies. So first, on paper plan is important to chalk-out a strategy.

Good luck to you and if any questions, many are there to help with advice.

krishnasastry

I have one thing to ask, are you amplifying your gene from a mixed genome (because you mentioned sludge sample), and your gene sequence is unknown. Say it is prokaryotic in orgin, are the primers you are using to amplify your gene is universal in nature. Are you sure that the amplification is taking place for the full length cds of your gene (i mean from start to end). If it is not so then you wont get any expressed product. And as everyone said, having sequence data of your amplicon before going for expression cloning is helpful. TA-clone it and get the sequence, use softwares to find the restriction sites on your target gene. use restriction enzyme sequences on your primer which dont not cut your target gene. Go for second amplification round, double digest for directional cloning and orientation ligate and express.

Try with the geneart selmless cloning along with vector construction. Invitrogen is having a limited vector series. You can try with the Eurofin and suggest your vector requirements from Novagen. You can also request for a customised vector too.

hi I clone my interest of gene in T/A vector and then digest with NdeI and XhoI. and gel purify my band, ligation with pET26 and transform. I see clones but clony- PCR results a smaller band. why?