Abstract
If you want to know whether zebrafish have a gene similar to human Gene X, what should you do? One simple approach would be to use UniGene in the NCBI database (National Center for Biotechnology Information; http://www.ncbi.nlm.nih.gov/unigene), which provides lists of orthologs (functionally equivalent genes). This means that if you enter the name of a gene into UniGene, for example Gene X, you get orthologs found in various organisms. Of course, this search is limited to information on Gene X and its orthologs catalogued in UniGene. In recent years, the gene data in UniGene have been accumulating exponentially.
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Some mRNAs, such as histone mRNAs, do not have poly(A). Rather, they form a stem-loop at their 3′ ends, which act like a poly(A) by stabilizing the mRNA and assisting with translation. It goes without saying that mRNAs without poly(A) cannot be amplified by 3′ RACE.
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Homologous arms as short as 40 bp would be OK for BAC recombineering. As such, it would be a good strategy to start with 40 bp. If this fails, increase the length of the homologous arms.
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EL250 has homologous recombinase and flippase integrated onto the chromosomes. The recombinase is induced by heat-shock and the flippase by arabinose.
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In the past, this often happened. However, recent vectors usually have at least eight RE sites in their MCS.
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As Pfu exerts very little polymerase activity at 25 °C (the temperature at which the ligation is carried out), this purification step may be omitted.
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Because pBlueScript II is small and there are many MCS, it is my favorite cloning vector. It is perfect for an intermediate vector when shuttling genes around.
This vector was originally developed by a company called Stratagene. The nomenclature of pBlueScript is quite confusing, which confused me quite a few times. I will clarify it for you.
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MCS are either KS or SK. If the MCS begins with SacI and ends with KpnI, it is called SK. If MCS begins with KpnI and ends with SacI, it is called KS.
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If the direction of the f1 origin is counterclockwise on the plasmid map, it is (+), and if clockwise, it is (−). +/− does not matter for the sake of cloning and thus there is no reason to be concerned. However, KS and SK should be distinguished clearly. Otherwise, you might end up with a mishap later on.
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An expert does not necessarily have to have enormous knowledge about cloning. You can ask for advice from your colleagues sitting right next to you. Even if your colleagues do not have as much knowledge as you have, she/he can help you pull it off. Or while talking with your colleagues, a light bulb may come on in your head.
- 8.
NLS stands for nuclear localization sequence. The protein with the NLS is targeted to the cell nucleus.
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Choi, SY., Ro, H., Yi, H. (2019). The Last Steps of Cloning. In: DNA Cloning: A Hands-on Approach. Springer, Dordrecht. https://doi.org/10.1007/978-94-024-1662-6_5
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DOI: https://doi.org/10.1007/978-94-024-1662-6_5
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