TOPO is costly... why you cloned in that instead of TA vector... Also if you cant get correct sequence then you are wasting the topo vector. If you cloned the full length sequence in TOPO, then its ok that you may use that in future for doing gateway cloning.

why you waste time on cloning and sequencing again and again..

Thanks for the suggestions. first of all i have used TOPO TA cloning vector (Invitrogen) and transformed it into DH5 cells. Also as i have said, i have conducted sequencing to validate my sequence (both full-length and partial). My objective is to produce recombinant protein using pET expression vector and IPTG induction of BL21-DE cells. But before conducting the same, i need to digest my insert in TA cloning vector with restriction enzymes. (in my case BamHI and HindIII). But after digestion, iam not getting the expected size, but a little higher than the expected size.

My insert is showing the correct size after conducting the PCR, though i dont know whether i can use the purified band after PCR as my insert. Please suggest.

The pCR2.1-TOPO vector also contains BamHI and HindIII sites about 40 and 60 bp upstream of the TA insertion site. Maybe one of the restriction sites in your insert is not cut but the site in the vector instead. Obviously, the surplus fragment size would only be 40 or 60 bp (instead of 100 bp), but who can estimate fragment sizes so exactly from an agarose gel?

I agree With Mr. radauer.

You can also use your PCR product if you amplify it with proofreading taq. For that you have to purify your pcr product on column if there is no parasital band or on gel if there is. The problem is that PCR product digestion is more difficult and as to be overnight to be really efficient if restriction site is at the end of the insert. Also ensure that you have more than 5 nt between the end of the insert and the restriction site. I did it and it works well.

Thanks for the suggestions. I think you are suggesting me to use my PCR band after gel purification as the insert for further restriction digestion (TOPO-insert) and after digesting pET vector-ligating both pET and insert for transformation and IPTG induction of the protein in BL21-DE3 cell lines. Normally iam conducting digestion with fermentas enzymes for a duration of 90 minutes. I think you are suggesting me overnight digestion. Please suggest.

My suggestion is the following plan :

1- TA cloning and sequencing verification on TOPO vector.

2-amplify by PCR from the plasmid PCR TOPO with primer including restriction site you need (with more than 5nt between ends and restriction site)

3-migration of 5µl of the PCR product

4- if no parasital band just pass on column, if parasital band gel purify all your product on low melting agarose.

5- prepare your mix digestion

6-digest overnight (i never use fermentas enzyme but for pcr product i always dgest overnight)

7-purify on gel the digestion

8- Digest your target (pET if i understand) vector and make CIP to ensure no religation

9-ligate on 1:5 ratio

10 transform

12 screen your procut!

That's my suggestion

Hope it will work

Thanks Dr. Halgand. To summarize your generous plan-

After verification and validating my sequence, i need to run PCR with expression primers (inclusive of 5'-BamHI and 3'-Hind III). Then, i should run 1% Agarose gel electrophoresis and check for band of expected size. The expected size band can then be gel extracted and purified. The purified product has to be digested overnight (as its the PCR product) and gel extracted and purified. Then the pET vector can be digested. The digests (insert with pET) can be ligated overnight (16hrs) and checked by running PCR with T7 primers. This can be followed up with transformation in BL21 cells (IPTG induction). Hope its ok. Please let me know any discrepancy. Also please let me know in detail step 8 (make CIP to ensure no religation). Thanks again.

that's it! do 100µl of pcr product to have the more pcr product you can because with all the purification you will loose a lot of product...

the CIP is just phophatase to ensure that your plasmid pET won't religate on himself without the insert...

Good luck!

And i think you'd better transform your ligation product and plate your bacteria then pick the colony on pcr screening than running pcr directly on your ligation product.... But as you want!

Dear Bharat, I just came upon this posting. My take on this is that your digestion of the pCR clone is the problem. It is possible that one of your enzymes is not working inthe buffer system that you are using OR that the restriction sites that you included on the PCR product are not cutting and the product you see is simply the result of digestion of the sites on the plasmid. I would suggest that in the first instance you test this by doing single BamHI and HindIII digests in the buffer you have been using for the double digest.

If one of the enzymes doesn't digest your clone, then you should look at changing the double digest buffer or doing the digests one at a time. This should release the smaller insert that you want to work with.

If both enzymes work, as detected either by linearising the plasmid or by releasing the insert (this depends on the order of the sites on the vector and you insert in the pCR clone) it suggests there is a problem with the sites added to your PCR product.

In this case it may well be that these sites will also be resistant to digestion in any PCR product you amplify from this clone. I would suggest that you CAREFULLY examine the squence of your insert in the pCR vector - especially at its ends where the restriction sites have been added. If the sites are clearly intact it may be easier/better/more productive to simply digest the pCR clone DNA with a large amount of enzyme overnight - taking care to work in a buffer that is compatible with both enzymes.

Thanks once again. Dear Dr. Halgand, PCR with the ligation product (pET vector with insert) is to check the sucess of the ligation product. For this iam using pET vector forward and reverse primer (T7). Alternatively we can use vector forward primer(T7) and gene specific reverse primer or gene specific forward primer and vector reverse primer (T7). This i hope can validate our construct towards transformation. I need to ask though, that as i will be verifying my reading frame by sequencing, is it better to transform into non-expression host as DH5-alpha and select for the white colonies as pET lacks lacZ gene for blue-white colonies. After confirming the sequence, transformation can be conducted in a expression host as BL21-DE3 cell lines and the target protein can be optimized by IPTG induction. Please comment.

Dear Dr. George, i have tried sequential double digestion as well with NEB enzymes (BamHI and HindIII) apart from fermentas enzymes, but i could not get my insert at the expected size, though PCR of the insert with expression primers gave the expected size. Do you suggest for overnight digestion.