Hi Naghmeh,

It helps to have primer design software to help work primer Tms and avoid repetitive sequences that might dimerise or form hairpins - but it is not necessary. You could try primer3 (its and online resource).

Making sure your gene sequence remains in frame with any other elements of your construct, simply add the 6base recognition sequence of XbaI or XhoI to the 5` end of the gene specific forward or reverse primers. To get efficient cleavage close to the end of the PCR amplicon you will need to add several more bases to the 5` of the enzyme cleavage sequence. The appropriate number for efficient cleavage of certain restriction enzymes can be found here:

https://www.neb.com/tools-and-resources/usage-guidelines/cleavage-close-to-the-end-of-dna-fragments

... so for XbaI and XhoI you should really add an additional 3-4 bases to the 5` end.

The resulting PCR amplicon will then have restriction enzyme cleavage sites at the 5` and 3` for insertion into your construct. Good luck,

Pete

Hi Naghmeh,

It helps to have primer design software to help work primer Tms and avoid repetitive sequences that might dimerise or form hairpins - but it is not necessary. You could try primer3 (its and online resource).

Making sure your gene sequence remains in frame with any other elements of your construct, simply add the 6base recognition sequence of XbaI or XhoI to the 5` end of the gene specific forward or reverse primers. To get efficient cleavage close to the end of the PCR amplicon you will need to add several more bases to the 5` of the enzyme cleavage sequence. The appropriate number for efficient cleavage of certain restriction enzymes can be found here:

https://www.neb.com/tools-and-resources/usage-guidelines/cleavage-close-to-the-end-of-dna-fragments

... so for XbaI and XhoI you should really add an additional 3-4 bases to the 5` end.

The resulting PCR amplicon will then have restriction enzyme cleavage sites at the 5` and 3` for insertion into your construct. Good luck,

Pete

Thanks Peter,

Do I need a mutation Kit? or it is only matter of primer designing.

Do you know any protocol?

Naghmeh,

Peter explained it very well. Here is another way of doing this:

You can always "blunt ligate" your insert into your construct. Here is how you can do this:

1) PCR amplify your fragment by a high fidelity polymerase (Accuprime, Phusion, Q5, etc..). Using these enzymes, the PCR product should be blunt. If you use Taq polymerase, the product will have an A overhang. In that case you need to blunt the ends by Klenow fragment (or blunting enzyme of your choice).

2) Phosphorylate the ends of your amplified insert using PNK (this will add 5' phosphate group to your fragment, which is needed for ligation).

3) Open your construct by restriction digestion at your desired site.

4) Blunt the ends of the constructs by Klenow fragment.

5) de-phosphorylate the ends of the construct using CIP (this will prevent re-circularization of the plasmid).

6) Mix the construct and the insert in 1:3 molar ratio and ligate using Quick ligase.

7) Transform

8) screen for correct insertion.

If you do blunt ligation a sI explained, your insert will be ligated either in the correct orientation or inverted orientation. You need to check this by sequencing or restriction enzyme digestion.

What I explained can be done in one day.

Hello again Naghmeh,

You will not need a mutagenesis kit if you only want to add restriction enzyme sites to your insert... However, if those sites are not present in the vector into which you want clone that insert, then you might need to mutagenise that vector. If this is the case you should first try to find compatible restriction enzyme sites (eg dont use XbaI and XhoI use two others).

Now that I re-read your question - it seems it is the case that the restriction enzyme sites are not at the end of your current construct. In which case try agilent's QuikChange mutagenesis kit:

http://www.genomics.agilent.com/en/Site-Directed-Mutagenesis/QuikChange-Lightning/;jsessionid=K1hzT1sfzxvynzH0fT3pv86N2xcmpnkGyGLWNtw7vM1hFSCTGQcQ!-341169411?cid=AG-PT-175&tabId=AG-PR-1162

The protocol they provide is quite good. The tricky part is designing the primer to insert the enzyme site you want... and on that same site is a "Primer Designer Program" that will help you get your primers just right (you will have to create a login to use).

If at all possible you should re-sequence your construct when you're done. PCR can sometimes introduce errors...

Pete

Hi NA: Overlap extension PCR would be the best way to ligate with or without the RE site. This much easier instead of RE digestion, purification etc. Just check the below link to have an idea about this method. http://en.wikipedia.org/wiki/Overlap_extension_polymerase_chain_reaction If needed, I will provide the steps to be followed. Need to know if the RE sites are really needed or it is just for the purpose of insertion. Good luck. - S. Kumar

Here is a low-tech way of doing this if reading frame or the size is of no concern: 1. cut your gene out of your construct with any two enzymes that flank it 2. Blunt-end your fragment 3. Ligate into EcoRV site of pBluescript (or analog). You can add EcoRV into ligation reaction to prevent vector from ligating to itself (if you choose to do so, watch for EcoRV sites in the insert!). Since EcoRV site in the pBluescript is located between Xba and Xho sites, all you have to do is to choose correct orientation if it is important. The advantage of this approach is that it does not involve PCR, and therefore there is no need to sequence your gene after amplification.

Hi Kaveh,

Actually, I do not have any restriction site in intrested place to cut it. In intrested place of plasmid, I would like to introdue two restriction sites, because I want to insert new sequence of fusion tag there. therfore, I need two restriction site in that place before fusion tag sequence insertion.

Now, I got new idea. Maybe, I can also insert the fusion tag sequence without restriction site introduction. do you think, is it possible at all?

i want to introduce two restriction sites at both the ends of an ORF within a circular genome. Can i have suggestions please?