Difference between revisions of "Part:BBa K191004:Design"

Revision as of 16:26, 21 October 2009

TRP promoter - RBS - RFP - Term

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal SpeI site found at 43
Illegal SpeI site found at 51
12
INCOMPATIBLE WITH RFC[12]
Unknown
21
COMPATIBLE WITH RFC[21]
23
INCOMPATIBLE WITH RFC[23]
Illegal SpeI site found at 43
Illegal SpeI site found at 51
25
INCOMPATIBLE WITH RFC[25]
Illegal SpeI site found at 43
Illegal SpeI site found at 51
Illegal AgeI site found at 666
Illegal AgeI site found at 778
1000
COMPATIBLE WITH RFC[1000]

This BioBrick was synthesized using the protocol we developed with the Klenow fragment([http://2009.igem.org/wiki/index.php?title=Team:EPF-Lausanne/Protocols/Klenow complete protocol]).
To do this, two long primers which together contained the sequence for the Trp promoter were ordered:

Forward primer: 5'-gtttcttcgaattcgcggccgcttctagagtaatcatcgaactagttaactagtacgcaag-3' Reverse primer: 5'-gctagcgaacttgcgtactagttaactagttcgatg-3'

These primers could self-anneal in the reaction mix, and the second strands that were missing at the extremities of the primers were fully synthesized using the Klenow fragment, instead of a classic extension using the TAQ DNA Polymerase.
Double strand Trp promoter was cut with E and S to be ligated into plasmid with RFP gene (BBa_I13507).

@@ Line 6: / Line 6: @@
 ===Design Notes===
-* Trp promoter was added to plasmid containing part <partinfo>BBa_I13507</partinfo> from iGEM kit using [http://2009.igem.org/wiki/index.php?title=Team:EPF-Lausanne/Protocols/Klenow Klenow protocol].
+* This BioBrick was synthesized using the protocol we developed with the Klenow fragment([http://2009.igem.org/wiki/index.php?title=Team:EPF-Lausanne/Protocols/Klenow complete protocol]).
-* Trp promoter as PCR products in Klenow protocol was cut with E and S to be ligated into plasmid with RFP gene (<partinfo>BBa_I13507</partinfo>), which was also cut with E and S.
+* To do this, two long primers which together contained the sequence for the Trp promoter were ordered:
+Forward primer: 5'-gtttcttcgaattcgcggccgcttctagagtaatcatcgaactagttaactagtacgcaag-3'
+Reverse primer: 5'-gctagcgaacttgcgtactagttaactagttcgatg-3'
+* These primers could self-anneal in the reaction mix, and the second strands that were missing at the extremities of the primers were fully synthesized using the Klenow fragment, instead of a classic extension using the TAQ DNA Polymerase.
+* Double strand Trp promoter was cut with E and S to be ligated into plasmid with RFP gene (<partinfo>BBa_I13507</partinfo>).
 ===Source===