Dear friends
Im trying to clone and express the riboflavin biosynthesis operon in E. coli. There are 4 overlapping genes and each contains it start and stop codon, and the total length of this is 8 kb. Is it possible to amplify 8 kb using Pfu?. Also is is possible to clone the operon in pET30 plasmid and express in E. coliBL21?
Please help
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#2
Adrian K
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Posted 08 September 2010 - 03:04 AM
Hi, for your first part of question, I had used novagen KOD hotstart and cloned a 9kb genes before. If your Pfu can't make it you shall try KOD hotstart as It also have the proof reading capability.
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"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong
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..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...
"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong
"It's all just DNA. Do it."---phage434
#3
HomeBrew
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Posted 08 September 2010 - 03:07 AM
I don't have too much experience with Pfu, but 8 kb is not that big -- I cloned a 15 kb operon using Hi-Fi Taq -- so it's possible. I don't understand why you're using a his-tag vector/strain system for this, but the PCR product is able to be cloned into any appropriately prepared vector. Whether it will be expressed in any given recipient strain of E. coli is dependent on whether it'll be toxic to the cell.
