I will highly appreciate it, if anybody could send me their experiments about Linker, vector and special attention during the design of primers. Because finally we want to fuse two gene and expressed those with linker. Thank you
Depending on the funds you have it is eeasier if you order a gene construct for example to Genscript.
Hi Abdollah
There are many factors involved in cloning of a gene into a vector.
Primers (reverse and forward) should must have close melting temperatures (up to 5 degree) to avoid difficulty during PCR.
The length should not be too long or too short. Ideal length should be in between 20 to 40 nucleotides.
The primers sequence should end with G or C nucleotide bases to have firm bonding to template during annealing.
The restriction site chosen should have zero cutter for the gene of your interest. Use NEB cutter to check the zero cutter restriction enzymes.
TRy to calculate the tm value of primers mannualy to avoid errors. Check for primer complement possibilities before finalising your primers.
Once you get successful pcr results, you can go for cloning. You should have enough concentration of linker and vector to have successful digestion and ligation. We use 1:3 ratio for vector:linker ligation mixture. Try to have at least 100ng of both vector and linker after restriction digestion to raise the possiblity of successful ligation. Use normal transformation protocol using antibiotic as selection pressure.
I hope, It will help you somehow.
Good luck.
Hi. you will have many options. 1. synthesis of your fusion gene 2. conventional method ( isolate your fragment with PCR and then ligate them to each other (same as my previous work I ligated a signal sequence , an intron an my intrested gene to each other + promoter)). 3. overlap PCR if it is possible. 4. gibson assembely method that created for this purpose.
i hope it will help you
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