(New page: === Construction details === pME-MCS was made by PCR of the pBSII SK+ multiple cloning site, from M13F to M13R primers, followed by a BP reaction with pDONR221. Sequencing confirms that al...) |
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pME-MCS was made by PCR of the pBSII SK+ multiple cloning site, from M13F to M13R primers, followed by a BP reaction with pDONR221. Sequencing confirms that all bases of the insert are correct. | pME-MCS was made by PCR of the pBSII SK+ multiple cloning site, from M13F to M13R primers, followed by a BP reaction with pDONR221. Sequencing confirms that all bases of the insert are correct. | ||
| − | Since the insert contains the entire M13F sequence, | + | Since the insert contains the entire M13F sequence, and the 3' end of the M13R sequence, M13F will definitely not work for sequencing clones made from pME-MCS (it will bind both in the vector backbone and in the insert), and M13R will work poorly (it will bind perfectly to the vector backbone and weakly in the insert). |
The following sites from the Bluescript MCS remain unique in pME-MCS: | The following sites from the Bluescript MCS remain unique in pME-MCS: | ||
Revision as of 23:32, 27 September 2007
Construction details
pME-MCS was made by PCR of the pBSII SK+ multiple cloning site, from M13F to M13R primers, followed by a BP reaction with pDONR221. Sequencing confirms that all bases of the insert are correct.
Since the insert contains the entire M13F sequence, and the 3' end of the M13R sequence, M13F will definitely not work for sequencing clones made from pME-MCS (it will bind both in the vector backbone and in the insert), and M13R will work poorly (it will bind perfectly to the vector backbone and weakly in the insert).
The following sites from the Bluescript MCS remain unique in pME-MCS:
KpnI-XhoI-SalI-Bsp106-HindIII-EcoRI-PstI-SmaI-BamHI-SpeI-XbaI-NotI-XmaIII-SacII-BstXI-SacI
Crudely annotated sequence
FASTA file with the full-length sequence as well as sequences of individual components:
pME-MCS sequence
Crude map
Screenshot from Sequencher showing locations of components:
