Fusion protein from PCR - construct is reversed.

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cts123
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Fusion protein from PCR - construct is reversed.

Post by cts123 » Fri Apr 16, 2010 11:45 pm

Hello,

I've been working on creating a fluorescent fusion protein using PCR to join up three segments. We sent it for sequencing and it's come back as precisely the reverse to what it should be. Has anybody got any ideas on what might have happened?

Many thanks,

cts

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JackBean
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Post by JackBean » Sat Apr 17, 2010 7:59 am

what do you mean by reversed? If your fragments were ab, cd and ef and you want to put it into abcdef, what was your result?
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.

cts123
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Post by cts123 » Sat Apr 17, 2010 4:55 pm

Sorry, I didn't make that very clear. It's basically gone efcdab.

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JackBean
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Post by JackBean » Sat Apr 17, 2010 7:15 pm

I see, were your fusion parts enough different?
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.

cts123
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Post by cts123 » Sat Apr 17, 2010 11:47 pm

As far as I'm aware they were fairly different. At first I wondered if there were any false priming sites, but we did check beforehand and none appeared significant. I'm just puzzled really!

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JackBean
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Post by JackBean » Sun Apr 18, 2010 11:52 am

interesting. I guess you did it in one step PCR, right? What about to fuse first two parts and than add the other.
Or you could fuse A with B; B with C and let some microorganism to fuse AB with BC for you :)
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.

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MrMistery
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Post by MrMistery » Mon Apr 19, 2010 12:51 am

can you post your exact protocol here so I can get a better picture?
"As a biologist, I firmly believe that when you're dead, you're dead. Except for what you live behind in history. That's the only afterlife" - J. Craig Venter

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