Hello everyone!
I am a Gibson Assembly beginner. I just got the sequencing results back from my cloning and they are perfect except for one thing- I have an extra histidine (7x). I made the primer design using Snap gene and did the ainulation in silico and there only six.
Any idea of how this could happen? Is a 7x histidine tag still useful?
thanks!
hi
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Although it isn't commonplace, a 7x His tag should still be functional. In conventional tags, while 6x His is the most common, people have used 8x His, 10x His, 12x His, and in some cases this does increase binding affinity / specificity to Ni2+ or other similar affinity resins.
ie - https://www.researchgate.net/post/Why_do_we_mostly_use_only_6xHis_Tag_to_purify_Proteins_Not_8xHis_Or_10xHis
Try expressing and purifying it - what is the worst that could happen?
I should stress I'm not a Gibson expert, so the only thing I can suggest for the earlier part of your question would be to cover the region of the His tag with a single long primer that goes beyond the His- residues at the 3' end so you shouldn't get PCR stuttering during the amplification step.
Good luck! Robin
Does the His-tag-coding region reside within one of the primers (sounds like)? Then it could be that just the primer is wrong, this happens sometimes during synthesis.
How many clones did you sequence yet? And what polymerase are you using for fragment generation? Maybe try one with proofreading activity if you didn't yet (phusion, Q5, KOD, or taq/pfu, taq/vent mixes will all work) If there were problems with the similar codons, this should result in some clones with additional HIS-codons but not all.
But in general, I agree with Robin; just try it, there is no reason it shouldn't work.
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