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Cloning big fragment into smaller vector


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#1 miss montana

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Posted 17 September 2009 - 08:05 AM

Hi Everyone,

I'm trying to subclone a cDNA fragment from a retroviral vector into a pcDNA3-FLAG vector. I've not had much success. I've cut my insert out of the original vector (NOTE: the original vector is about 6kb and my insert is 7kb, making it hard to get the cDNA only), linearized the FLAG vector, gel purified, ligated overnight and transformed in XL10's. The first time I did it, I got some (maybe 35) colonies from my cDNA/FLAG vector ligation reaction. However, most of the bugs contain the original vector or, if they do contain the insert, the DNA yield is really low, about 60 ug/ml, and the quality is crummy.

I tried it again and I got no colonies after transforming the ligation reaction product. Any suggestions?

Thanks!

#2 Warren

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Posted 18 September 2009 - 08:51 AM

Hi Everyone,

I'm trying to subclone a cDNA fragment from a retroviral vector into a pcDNA3-FLAG vector. I've not had much success. I've cut my insert out of the original vector (NOTE: the original vector is about 6kb and my insert is 7kb, making it hard to get the cDNA only), linearized the FLAG vector, gel purified, ligated overnight and transformed in XL10's. The first time I did it, I got some (maybe 35) colonies from my cDNA/FLAG vector ligation reaction. However, most of the bugs contain the original vector or, if they do contain the insert, the DNA yield is really low, about 60 ug/ml, and the quality is crummy.

I tried it again and I got no colonies after transforming the ligation reaction product. Any suggestions?

Thanks!

One thing you can do is find an additional enzyme that cuts somewhere in the middle of the original vector but not your insert, so that the band you wish to cut out is more distinctive on the gel....w...

#3 caren

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Posted 24 September 2009 - 09:56 PM

Are you run gel for check ligation product?
What cell that you transformed?
Are you legate Blunt-end or Sticky-end?
The problem may be from your vector or your insert not match.

#4 miss montana

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Posted 25 September 2009 - 12:15 PM

I have not run a gel to check for a ligation product. I don't have all that much of it so I've never tried. I've speced it and I do have DNA.

I'm using XL-10s for the transformation. They're high efficiency and take up big plasmids.

I'm both my insert and recipient vector are cut with EcoRI, so the ends not matching isn't a problem.

Are you run gel for check ligation product?
What cell that you transformed?
Are you legate Blunt-end or Sticky-end?
The problem may be from your vector or your insert not match.






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