Hi Ranjana,

1.

How about using an enzyme that allows complementary binding, even if it is another restriction enzyme? Please refer to the following site.

https://international.neb.com/tools-and-resources/selection-charts/compatible-cohesive-ends-and-generation-of-new-restriction- sites

For example, for NcoI, BspHI, FatI, and PciI can bind.

For BglII, BamHI can be used.

For SpeI, AvrII, NheI, StyI, and XbaI can bind.

2.

Insert an arbitrary restriction enzyme site between NcoI, SpeI, and BglIII using two oligomers of about 20 bp each.

3. make oligomers of about 20 bp complementary to NcoI, SpeI, and BglII while providing unique restriction enzyme sites on both sides of the gene to be inserted, cross-link the insert with pCAMBIA1302, and ligate.

I can share the protocol if you have questions about the detailed methods for number 2 and 3.

Best,

Narumi Uno suggests some good solutions to your problem. A couple of other possibilities are:

Use a type IIs restriction enzyme. These cut outside the recognition sequence, so you can create a linker with the enzyme recognition site and then the sequence for your cloning. This method is commonly used for "Golden Gate" cloning. The favourite enzymes are BsaI and BsmBI, but there are lots of others that also work. (https://www.neb.com/applications/cloning-and-synthetic-biology/dna-assembly-and-cloning/golden-gate-assembly has more information, other suppliers obviously sell the same reagents!)

Often the best way today is to use a gene synthesis company. This allows you to choose exactly what sequence goes into your gene. Depending on the length of the gene it can often work out cheaper than PCR.

Good luck! Nic

I would suggest you to try ELIC, (Koskela 2015), it doesn't requiere digestion of PCR fragment and neither extra enzymes, you will only need to order new primers. The method is quite flexible and I have tried using a different strain of E.coli (DH5a) instead of XLBlue and it works perfectly. Also, I have used a shorter homologous ends (around 15-18 bp and no problems).

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