From Tol2Kit
(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...) |
|||
| (2 intermediate revisions by the same user not shown) | |||
| Line 1: | Line 1: | ||
| − | + | === 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. | + | 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:<br> | ||
| + | [[p5E-MCS Genbank]] | ||
| − | |||
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 === | |
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:
