I designed primer to clone the target gene with RBS, his tag.
I want to confirm below:
1. if Is it okay that extra sequence longer than target gene part?? Targeted gene part is 18bp long
But including rbs histtag part is about 41bp
I concerned if it's is too big size
2. I designed as RBS located after his tag sequence
But should I put RBS first and. His tag sequence later????
Please kindly give me advice
Thank you in advance!!!
Hi Su Yeong Ma ,
It's quite normal to design a primer with 40 bp in length. But it is important to make sure this length does not result in primer dimer ( self or hetero dimer), and any sort of hairpin. you can check it on OligoAnalyzer server from IDT.(Set the Mg Concentration on 1.5mM).We use simillar primers with same length but for other puposes. Our primer contain a tail about 15 bp and a target region about 25 bp.
So definatuly you you will not have any problem with that if its thermodynamic features are OK.
Best,
Ali
Dear Su Yeong Ma
you can certanilly add the his-tag and RBS in the primer if those are not present in the vector but if you would like to express the his-tag in your construct, the His-tag sequence have to be located after (and not before) the RBS and it have to start with an ATG codon, Since the protein codification begin from the first ATG located after the RBS.
best
Manuele
Thank you all for your kind and helpful question!!
I have replaced its order and i will check primer again!
And if it is okay, i want to confirm one more i designed as three seperated( hyphens indicate each different restriction site according MCS of vector)
Promoter-(300bp)RBS-(9bp space)- Signal peptide-histag&target gene
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