I was interested in using synthesized DNA for cloning. I am still used to the PCR based cloning. Any suggestions or recomendations before starting this process? Any good companies?
I have used MWG Operon. It took 8-10 business days and cost me about 250 dollars for a 760bp gene. The pro about using synthesized DNA is you can flank restriction sites at the end of your gene but look out for the presence of the same restriction sites on the plasmid vector that they sent you. If you cannot avoid this problem, you might have to do gel extraction and that can be a pain. PCR based cloning can be cheaper but it can come up with problems such as restriction sites being on the end of the gene (in this case restriction digestion can be difficult), avoiding introns (if you are dealing with eukaryotic DNA but cDNA would not be a problem), designing primers, etc. If you decide to use synthesized DNA, make sure you clone whatever they sent you first and don't do restriction digestion immediately.
I've done a little bit with using synthesized DNA and there are upsides and downsides to it. The big downside is the cost, though this is dropping as technology improves. As Nanda has said, the real benefit is being able to synthesize exactly the sequence you want with restriction sites where you want them. Also, some of the gene synthesis companies with do the sub-cloning for a small fee (compared to the overall price) which will save you the aggravation of doing it yourself (if the gene is tricky to clone).
Yes, for the first time I have used synthesized DNA of whole SIV viral genome for cloning and subsequent production of infectious SIV particles. Although it is expensive but the need for synthesized DNA for cloning depends upon your project requirements. Many people synthesize DNA flanking specific restriction sites for easy cloning. I used synthesized DNA of viral genome to understand whether synthesized DNA could produce infectious viral particles. Indeed it can! First thing before synthesizing DNA, is to have a complete cloning strategy including the unique restriction sites which you will use further to sub clone the DNA in the vector of your interest. We synthesized the whole viral genome from " Blue Heron Biotechnology". Although it is a bit expensive but they are quick and reliable. They also give you a stab culture plus purified plasmid with the cloning details they used. So the DNA synthesis for cloning is a bit expensive but you can achieve your target quicker without much of a hassle of PCR amplification and purification.
Yes, I have used Genescript for synthesizing a receptor of about 3kb and the sequencing results showed perfect sequence. Since it was difficult to get this gene as a PCR product, the cost to synthesize outweighed the struggle to PCR from cDNA.
Thanks Clement. I used origene many times and Blue Heron Biotechnology is what promted me to ask the question as I was looking for one of my genes with origena dn they did not have it so it was suggested to synthesize it.
Thanks Muralidharan. I totally agree, sometimes people do not factor in the salary of the person doing the job as well and the time. I did the calculations in my PhD and it was about the same coast if not chaeper to by ready made clones.
Cloning with synthesized appear to be a good alternative to conventional gene cloning. The major factor to put into consideration may be the codon usage. Codons that are common in some species may be rare in others and so expression can become limited.
I successfuly used a synthetic gene (2526 bp) to clone yeast and bacteria. This gene was synthesized by Sangon Bioengineering Company (Shanghai, China); GeneScript also can provide with optimized gene for cloning.
Hi - I have experience with GenScript and DNA2.0, the letter being the best for me. Here is the link to their website where you can find the details : https://www.dna20.com/services/gene-synthesis?gclid=COSGibmetLkCFYRP3godcVwAvg . They also have a very efficient optimization and rush order services.
I had GenScript (http://www.genscript.com/gene_synthesis.html) synthesize an ~2,000 bp gene with codons optimized for inset cells, plus lots of modifications to enhance RNA stability. The sequenced and cloned gene, in their plasmid, cost $730. I highly recommend them. I could have bought the gene in a plasmid and then used PCR to modify the 5' and 3' ends as needed and then clone it into my vector, but when the cost of reagents (enzymes/primers) and sequencing were factored in GenScript was more cost effective.
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