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EPF-Lausanne/4 August 2008
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The negative control (RFP with RhlR primer) did not work. The two first positives with Biobrick primers worked. However, the positive with RhlR primers failed. But this RhlR sequence was reported to be inconsistent, which could account for our trouble. | The negative control (RFP with RhlR primer) did not work. The two first positives with Biobrick primers worked. However, the positive with RhlR primers failed. But this RhlR sequence was reported to be inconsistent, which could account for our trouble. | ||
| - | We now proceed with the PCR up from the punched-out DNA. | + | We now proceed with the PCR up from the punched-out DNA, the same primer and DNA pairs are used, and in addition we try also with the Lac Promoter. |
| - | == | + | ==pLac == |
| - | We have checked the sequence of the Lac promoter used by Ron Weiss in his PHD1 plasmid by blasting the promoter part from iGEM sequence ([http://partsregistry.org/wiki/index.php?title=Part:BBa_R0010 Part R0010]) versus the complete plasmid sequence ([http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&val=59800245 Expression vector pHTSUB-105]). The high similarity gives us good confidence that the iGEM part would work as well. | + | We have checked the sequence of the Lac promoter used by Ron Weiss in his PHD1 plasmid by blasting the promoter part from iGEM sequence ([http://partsregistry.org/wiki/index.php?title=Part:BBa_R0010 Part R0010]) versus the complete plasmid sequence ([http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&val=59800245 Expression vector pHTSUB-105]). The high similarity gives us good confidence that the iGEM part would work as well. Since the PCR with Biobrick primers seems to work, and we don't have the part in swipe stock, we will try to get it from the Binder stock. We try a PCR on the same Biobricks as previously with the same primers, but using the DNA from the binder stock, plus pLac with the Biobrick primers. |
Latest revision as of 12:12, 7 August 2008
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Cell culture and miniprep
The DNA which was obtained on 01/08 by miniprep was put on a gel. The gel was clean and correct for the DNA with AmpR, but somehow unneat for the KanR/AmpRKanR ones. One which had been in very high concentration (300 ng/uL) seemed overloaded. The others seemed to contain genomic DNA, which could come from contamination or could be due to the use of spin columns from a different miniprep kit. We will in any case grow them out again with different Kan stock and proceed tomorrow with a digestion reaction, of shorter than overnight, to check the authenticity of the parts.
PCR
A PCR with the following primer-DNA sets was done :
| Primer | Biobrick |
RhlR |
| DNA | RhlR (I0466) |
RFP (E1010) |
The negative control (RFP with RhlR primer) did not work. The two first positives with Biobrick primers worked. However, the positive with RhlR primers failed. But this RhlR sequence was reported to be inconsistent, which could account for our trouble. We now proceed with the PCR up from the punched-out DNA, the same primer and DNA pairs are used, and in addition we try also with the Lac Promoter.
pLac
We have checked the sequence of the Lac promoter used by Ron Weiss in his PHD1 plasmid by blasting the promoter part from iGEM sequence ([http://partsregistry.org/wiki/index.php?title=Part:BBa_R0010 Part R0010]) versus the complete plasmid sequence ([http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&val=59800245 Expression vector pHTSUB-105]). The high similarity gives us good confidence that the iGEM part would work as well. Since the PCR with Biobrick primers seems to work, and we don't have the part in swipe stock, we will try to get it from the Binder stock. We try a PCR on the same Biobricks as previously with the same primers, but using the DNA from the binder stock, plus pLac with the Biobrick primers.
Score = 291 bits (151), Expect = 1e-75
Identities = 155/157 (98%), Gaps = 0/157 (0%) Strand=Plus/Plus
Query>044 ATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAA 103
Sbjct>2317 ATGAAGCTAGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAA 2376
Query>104 TGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTAT 163
Sbjct>2377 TGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTAT 2436
Query>164 GTTGTGTGGAATTGTGAGCGGATAACAATTTCACACA 200
Sbjct>2437 GTTGTGTGGAATTGTGAGCGGATAACAATTTCACACA 2473
CPU time: 0.05 user secs. 0.03 sys. secs 0.08 total secs.
