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(New page: '''Construction details'''<br> p5E-MCS was made by PCR of the pBSII SK+ multiple cloning site, from T7 to T3 primers, followed by a BP reaction. Sequencing confirms that all bases of the i...)
 
 
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'''Construction details'''<br>
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=== Construction details ===
p5E-MCS was made by PCR of the pBSII SK+ multiple cloning site, from T7 to T3 primers, followed by a BP reaction. Sequencing confirms that all bases of the insert are correct.
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p5E-MCS was made by PCR of the pBSII SK+ multiple cloning site, from T7 to T3 primers, followed by a BP reaction with pDONR P4-P1R. Sequencing confirms that all bases of the insert are correct.
 +
 
 +
M13F, M13R, and T3 should all work as sequencing primers.
 +
 
 +
Unfortunately, the backbone of P4-P1R contains recognition sites for several potentially useful restriction enzymes. The restriction sites from the Bluescript MCS that remain unique in p5E-MCS are:
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''Asp718-KpnI-DraII-XhoI-SalI-Bsp106-HindIII-SmaI-BamHI-SpeI-SacII-BstXI''
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=== Sequence ===
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Annotated sequence, Genbank format:<br>
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[[p5E-MCS Genbank]]
  
'''Crudely annotated sequence'''<br>
 
 
FASTA file with the full-length sequence as well as sequences of individual components:<br>
 
FASTA file with the full-length sequence as well as sequences of individual components:<br>
 
[[p5E-MCS sequence]]
 
[[p5E-MCS sequence]]
  
'''Crude map'''<br>
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=== Crude map ===
 
Screenshot from Sequencher showing locations of components:<br>
 
Screenshot from Sequencher showing locations of components:<br>
 
[[Image:p5E-MCS.png]]
 
[[Image:p5E-MCS.png]]

Latest revision as of 03:43, 19 December 2007

Construction details

p5E-MCS was made by PCR of the pBSII SK+ multiple cloning site, from T7 to T3 primers, followed by a BP reaction with pDONR P4-P1R. Sequencing confirms that all bases of the insert are correct.

M13F, M13R, and T3 should all work as sequencing primers.

Unfortunately, the backbone of P4-P1R contains recognition sites for several potentially useful restriction enzymes. The restriction sites from the Bluescript MCS that remain unique in p5E-MCS are:

Asp718-KpnI-DraII-XhoI-SalI-Bsp106-HindIII-SmaI-BamHI-SpeI-SacII-BstXI

Sequence

Annotated sequence, Genbank format:
p5E-MCS Genbank

FASTA file with the full-length sequence as well as sequences of individual components:
p5E-MCS sequence

Crude map

Screenshot from Sequencher showing locations of components:
P5E-MCS.png