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problem with gateway cloning: missing part of insert


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#1 taphrina

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Posted 13 September 2013 - 01:58 AM

Currently I am trying to clone an attB PCR product into gateway pDONR221. After performing the BP reaction I got some few clones which I checked by M13 PCR and they seemed to be positive. However, sequencing revealed that quite a substantial part of the gene is missing, i.e. starting with a PCR product of almost 600 bp, I end up with only a bit more than 300 bp inserted into the vector.

Subsequently I cloned the attB PCR product into pGEM, checked it by sequencing and got exactly what I expected: my 600 bp sequence with the attB sites on both ends. So I think it is a problem with the BP clonase reaction?

Has anyone encountered similar problems? Any ideas?



#2 Ahrenhase

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Posted 13 September 2013 - 02:36 PM

BLAST your attB sites against your insert once



#3 taphrina

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Posted 16 September 2013 - 07:20 AM

I checked this already, but I don't find anything similar to the attB sites within the insert






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