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Cloning to add FLAG tag at c terminus....a big mistake

Cloning to add FLAG tag at c terminus....a big mistake - Molecular Cloning Forum

Cloning to add FLAG tag at c terminus....a big mistake - DNA cloning forum. Discuss the cloning of plasmids, vectors, DNA, RNA, cDNA, proteins, and other molecules for expression, sub-cloning, and other methods. Also discuss digestion, ligation, transformation, plasmid preparation, cDNA library screening, and competent cells here.


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Old 03-03-2010, 01:29 PM
Pipette Filler
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Default Cloning to add FLAG tag at c terminus....a big mistake



Dear Friends,
I have done a huge blunder while cloning different mutants of my protein. I planned to have FLAG tag at c terminus therefore I desigend primer for pcr with flag tag sequence and restriction site at c terminus. Now the mistake i did was i forgot to add stop codon after FLAG tag. I have cloned my protein and its mutant (5 different mutants) in KPNI and BAMHI site of pcDNA3. I checked the plasmid sequence and i found there ia an internal stop codon in plasmid which adds 28 more amino acid after flag tag befor getting terminated. I don't know what should i do....again start cloning with right primer with stop codon or it is okay to have similar strecth of extra amino acid in all my mutants as far as my flag tag is working fine. Flag tag is working fine and i get positive signal on western blot for all my mutants. Please please help me...i have very less time left with for finishing my project...what should i do?


Thanks & Regards
Sandhya
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Old 03-03-2010, 06:41 PM
Pipette Filler
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Default Re: Cloning to add FLAG tag at c terminus....a big mistake

Quote:
Originally Posted by pcrsanju View Post
Dear Friends,
I have done a huge blunder while cloning different mutants of my protein. I planned to have FLAG tag at c terminus therefore I desigend primer for pcr with flag tag sequence and restriction site at c terminus. Now the mistake i did was i forgot to add stop codon after FLAG tag. I have cloned my protein and its mutant (5 different mutants) in KPNI and BAMHI site of pcDNA3. I checked the plasmid sequence and i found there ia an internal stop codon in plasmid which adds 28 more amino acid after flag tag befor getting terminated. I don't know what should i do....again start cloning with right primer with stop codon or it is okay to have similar strecth of extra amino acid in all my mutants as far as my flag tag is working fine. Flag tag is working fine and i get positive signal on western blot for all my mutants. Please please help me...i have very less time left with for finishing my project...what should i do?


Thanks & Regards
Sandhya
How much time is "very less time"? Honestly, I would design a new primer and start over. You could spend a lot of time and waste a lot of money and reagents collecting data which is artificial due to those 28 amino acids. I wouldn't risk it. It's fairly simple to clone it again and do it correctly.

Again, I think the best thing is to make a new PCR primer and start over.
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