5 replies to this topic

[Sally-]

Hello,
I want to which is the best strategy to insert an HA tag in the middle of the sequence of a gene. I can't put the Tag neither at C-ter nor at N-ter because it will interfere with the interactions of my protein. I've tried to insert the tag by PCR cloning but the primers were very long and made the PCR reaction too difficult.

Thank you!!

[T C]

Hey

U can choose 2 sites which are close to each other in the gene, take this fragment out and insert a synthetic oligo containing this region with the tag.

Hope this helps.

Best,
TC  

Sally-, on Jul 9 2009, 01:04 AM, said:

Hello,
I want to which is the best strategy to insert an HA tag in the middle of the sequence of a gene. I can't put the Tag neither at C-ter nor at N-ter because it will interfere with the interactions of my protein. I've tried to insert the tag by PCR cloning but the primers were very long and made the PCR reaction too difficult.

Thank you!!

[cellcounter]

Sally-, on Jul 8 2009, 10:34 AM, said:

Hello,
I want to which is the best strategy to insert an HA tag in the middle of the sequence of a gene. I can't put the Tag neither at C-ter nor at N-ter because it will interfere with the interactions of my protein. I've tried to insert the tag by PCR cloning but the primers were very long and made the PCR reaction too difficult.

Thank you!!

I think the best strategy would be site directed mutagenesis.

[bob1]

It's pretty big for SDM, I would make a HA tag with cut sites on each end, and ligate it into a complementary single cut site somewhere in the middle of your gene.

[T C]

Yeah.....this is even better......should have thought abt this

bob1, on Jul 10 2009, 06:39 AM, said:

It's pretty big for SDM, I would make a HA tag with cut sites on each end, and ligate it into a complementary single cut site somewhere in the middle of your gene.

[cellcounter]

bob1, on Jul 9 2009, 04:09 PM, said:

It's pretty big for SDM, I would make a HA tag with cut sites on each end, and ligate it into a complementary single cut site somewhere in the middle of your gene.

I disagree. It is pretty efficient at HA tag size, done quite frequently, and the quickest and easiest way to achieve this without cloning acrobatics or relaxing the exact location where you want to insert your tag.