I do a M.Sc. thesis about expression of recombinant protein in Hansenula polymorpha (syn. Pichia angusta). Since there is no commercial vector available, we have to construct it ourselves. I have to clone three genes from Hansenula genome (promoter, terminator, and HARS1), but in my project have many step to do before I can graduate.
My thesis plan: Strat with cloning genes and plasmid construction, transformation, selection the clone, expression, SDS-Western blot, ELISA, Real-time PCR, and see a virus-like particle.
I wish to graduate in 3 years, but I have 2 years left.
Could you suggest how I should synthesize these genes or clone by myself? Actually, I want to clone these genes by myself but I heard that this step is hard, and take very long time.
With my respect
Tatpong Boontawon
If you prepare and read up, the cloning step can be done under two weeks, considering you have to order your primers, and taking in account that you're learning. With some genes it is easy, with others hard, but you never know until you check. Biology is an experimental science, go and experiment :) For the future, Addgene is a good resource for the plasmids that were already made.
If you prepare and read up, the cloning step can be done under two weeks, considering you have to order your primers, and taking in account that you're learning. With some genes it is easy, with others hard, but you never know until you check. Biology is an experimental science, go and experiment :) For the future, Addgene is a good resource for the plasmids that were already made.
Hi
If you want, you have more time to do that. It is simple and attractive. I did it many times. Don't worry and do it yourself. I cloned 6 DNA fragments into 4 plasmids and then constructed 2 expression plasmid for the transformation of the common white mushroom. I did it for 14 times, at first it was terrible, but I did it. I did it for 3 weeks, but I hardly worked at 7 to 22. Ok, that was my lab history. Please be careful about selection of restriction enzyme don’t use REs with ATG recognition site after the promoter and before the translation start site. Be careful:)
Cloning is not hard, but like every multistep process, provides lots of opportunities for making time-costly beginner mistakes. If time is short, you have no expertise, and ESPECIALLY, if you have no one in your lab with enough expertise to help, I'd suggest going for gene synthesis, if your funding allows it. Many companies will gladly provide the gene already cloned in the vector of your choice. And if you're game for additional advice, do not design the constructs by yourself, but try to enlist the help of someone knowledgeable.
As some colleagues have already mentioned, ordering a synthesis service from a company always considering your particular requirements (expression host, etc.) is the fastest and more precise way to obtain the construct you need. However, it depends on the budget you have. Cloning is not necessary a very long process if you use Gateway recombination methods, but it is not exactly a cheap alternative either. Take the advice of the other colleagues for having a person with cloning expertise on your side. It is absolutely necessary for everybody who is at the beginning of his career. Good luck
If you want to learn, it is better to do it self. However, if your lab has money and you want to get the construct quickly, gene synthesis by a company is the better option.
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