adding amino acids - (Aug/07/2008 )

Dear all,

I hope someone can explain me how to add 5amino acids.
Because of cloning I deleted first five amino acids,but now I need to do cristalization and I need that five a.a.

I am looking forward for solution

Lets assume you have this sequence:
a 5' HindIII and a 3' EcoRI site - in between your ORF
______H__I__...T__H__E__R__E__
AAGCTTCACATC...ACCCACGAGCGGGAGGAATTC
now you want to add 5 aminoacids in betwwen HindIII and the first H (histidine): lets assume MARIA
First of all you need to backtranslate this into DNA: ATGGCGCGTATTGCG
This can be done online: Type in Maria and press the "Amino to DNA" button.

Now you need to design a primer like:
HindIII-newDNA----------oldDNA
______M__A__R__I__A__H__I__...
AAGCTTATGGCGCGTATTGCGCACATC...

Afterwards a round of PCR, cloning, sequencing ....

Codon usage might be an issue for the first codons ATG GCG CGT ATT GCG so you better check either using the graphical codon usage analyzer or you do the codon usage optimization using the online tool of my employer. You even could order an optimized synthetic gene solving all your problems in one go!

thanks a looot

Dear MrGene

That new DNA I dont have . so I need to creat somehow from nucleotides?Can You please explain these step.I understood everything,but I dont know how to make MARIA?

Thanks