hey guys, here is a new Molecular cloning master student,I know this is a broad questions, and I also know it's about the target gene and vector and choose right Endonuclease, my problem is, sometime the primer I designed were not working for PCR or the it's woorking for PCR but the recombinant plasmid which I get were not working for protein express, so here I'm asking is there any tricks or software or any method to design a primer?or u have any refenrence to recommend.... Thanks a lot!!!
https://www.addgene.org/protocols/pcr-cloning/
https://www.embl.de/pepcore/pepcore_services/cloning/pcr_strategy/primer_design/
I've had good success with primer 3 tool at NCBI. It also incorporates Blast search so you can immediately see if your primer is amplifying anything else from your organism or other organisms (for me this is relevant as I work with viruses so I always have additional genome to think about).
Also, it is always worth the time to play a bit with annelaing temperature if you don't get the desired results with your PCR. Lower it by 1-2 degrees if there's no amplicon or increase it if there are additional bands.
https://www.addgene.org/protocols/pcr-cloning/
https://www.embl.de/pepcore/pepcore_services/cloning/pcr_strategy/primer_design/
I've had good success with primer 3 tool at NCBI. It also incorporates Blast search so you can immediately see if your primer is amplifying anything else from your organism or other organisms (for me this is relevant as I work with viruses so I always have additional genome to think about).
Also, it is always worth the time to play a bit with annelaing temperature if you don't get the desired results with your PCR. Lower it by 1-2 degrees if there's no amplicon or increase it if there are additional bands.
There are many software but you have to use the recommended one by kits producer in use.
you can design primers for molecular biology using two approach
1. primer designing for particular known genes you can use primer 3
2.if you dont know the exact genes than you take many similar sequences from NCBI after that go for alignment using MEGA software. so can design the primers according to the consensus sequences present among the genes.
Regarding software I would definitely advise the use of Benchling for everything even only remotely related to DNA. It is a completely free of charge online tool suit with a plethora of tools for visualizing, editing and modifying nucleotide sequences (and by now even protein sequences). Tools for generating primers are included as well. It is very intuitive and comes with a lot of video and text tutorials. Great support, too. I discovered it once by chance and have been advising it to everyone ever since!
https://www.benchling.com
For cloning and expression purpose you can design the primers by several ways.
1. You can design the primers manually while doing this you have to take care of no of bases which should not be more than 30 including the restriction enzyme site (27 is optimum). GC content should not be more than 55%. Hairpin loop temperature should be less than 20 degrees. Sometimes it's difficult to do this manually, so you can download the Gene Runner software and put your gene sequence in the software and check for the different primer set. Finally, your primer should look like as:
GAG - enzyme restriction site- Gene sequence
where GAG is the sitting position for the restriction enzyme. Here you also have to check for the in-frame position of your gene in the selected vector.
2. Another option is Primer3 or the NCBI primer site.
In all cases, after designing the primer you should check the specificity of your primer by doing primer BLAST on NCBI specifically against your organism's genome.
If you face any other problem you are welcome to contact me.
Best of luck
Gagan
https://www.thermofisher.com/bd/en/home/products-and-services/product-types/primers-oligos-nucleotides/invitrogen-custom-dna-oligos/primer-design-tools.html
https://www.youtube.com/watch?v=tUyBwiyMsSU
These link may be helpful for u
Hello Dear
https://www.embl.de/pepcore/pepcore_services/cloning/pcr_strategy/primer_design/
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