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Sequencing Wrong Cloned Insert

Sequencing Wrong Cloned Insert - Molecular Cloning Forum

Sequencing Wrong Cloned Insert - 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 05-01-2011, 09:45 AM
Pipette Filler
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Default Sequencing Wrong Cloned Insert



Hey everyone! I hope I'm posting this in the correct forum!

I isolated the internal transcribed spacer region of a fern leaf and transformed into E. coli using a pCR4 TOPO vector. I isolated the PCR product again using EcoRI. My professor then prepared it and sent it off for sequencing.

When the sequences came back, I trimmed off the vector sequence still attached and ran a BLAST search to verify that I had fern. But when I ran the search, the sequence came back saying that it belonged to SPINACH!!! So surprising!

I have to write a lab report over this and I have no idea how fern turned into spinach. I read an article talking about contamination, but I have no idea how fern would come into contact with spinach. It was definitely a fern plant, and not spinach. Any ideas are welcomed! Thanks

Last edited by admin; 05-01-2011 at 11:20 AM.
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Old 05-01-2011, 11:21 AM
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Default Re: Sequencing Wrong Cloned Insert

Hi there
can you post your primers? can you also post your sequenced insert - ie the sequence?

Also when you did TOPO cloning, did you use pipettes that were not used by other people working on spinach?
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Old 05-01-2011, 09:01 PM
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Default Re: Sequencing Wrong Cloned Insert

The primers I used were ITS-2 and ITS-5m. And I don't know why I didn't get the sequence from my professor! Now I feel dumb! But I was trying to get the internal transcribed spacer region to do plant phylogeny.

Well I can't account for people that have used the pipetters before me, but I do know I used fresh tips everytime.

I also cannot account for the person who brought in the leaf. I brought in a dandelion leaf initially, but my transformation failed, so the professor assigned me the fern PCR product that was already linked to the TOPO vector.

Thanks so much for your help!

Last edited by Mandoozle; 05-01-2011 at 09:37 PM. Reason: clarification
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Old 05-01-2011, 09:29 PM
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Default Re: Sequencing Wrong Cloned Insert

But thanks for bringing the pipetter contamination up, I just asked my professor if spinach was prepared for sequencing at the same time as the fern.
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Old 05-02-2011, 01:30 AM
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Default Re: Sequencing Wrong Cloned Insert

Just got the message from the prof. A student tried to isolate DNA from spinach this semester, but the PCR failed to amplify the internal transcribed spacer. They went ahead and tried to transform it, but that didn't work.
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