Dear Researcher,
I am characterizing a domain of an arabidopsis gene and in order to do that, i am making internal gene deletion of around 300bp region, located inside of the gene of interest. Since i am using conventional method in my laboratory, may i know is it acceptable if i replace the domain of interest (~300bp) with a single restriction enzyme site (for eg BamH1) and analyse these deletion construct for its functions? In detail, I will design two sets of primers spanning front region and back region excluding the domain of interest, At the 3'end of my front primer and 5' end of back primer i introduced BamH1 restriction site. After the amplification, both front and back fragment is ligated and the resulting construct will be sequenced to make sure no amino acid shift.
I will appreciate if there is any reference/publication attached for these method. Thank you!!
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Any advice on the methodology of internal domain deletion analysis? - ResearchGate. Available from: https://www.researchgate.net/post/Any_advice_on_the_methodology_of_internal_domain_deletion_analysis [accessed Jan 12, 2016].
Just make sure your introduced restriction site is unique (not present in the rest of your target template). The alternative is just to have your desired insert(s) synthesized, if it's not too big. The biggest concern is keeping the reading frame intact, as you already mentioned.
Hi,
I suggest you to try two methods which do not need to use the ligase.
1. overlap PCR
2. Gibson assmebly, as the following picture shows:
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