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Team:Tsinghua-E/Notebook
From 2013.igem.org
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We cloned dnaQ gene from E. Coli BL21(DE3) by PCR and utilized overlap extension PCR to introduce mutagenesis in dnaQ by the following protocol. | We cloned dnaQ gene from E. Coli BL21(DE3) by PCR and utilized overlap extension PCR to introduce mutagenesis in dnaQ by the following protocol. | ||
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| + | Firstly, we used E. Coli BL21 (DE3) strain as template to amplify dnaQ gene by PCR with mut-F and mut-R primers, respectively. After purification of PCR product (the 〖E.Z.N.A.〗^TM Gel Extraction Kit, produced by Omega Bio-tek), it was used as template to introduce point mutation by two parallel PCRs with the following two sets of primers: mut-L73W-F and mut-L73W-R (generating L73W); mut-A164V-F and mut-A164V-R (generating A164V). The three PCR products were gel-purified and combined by 5ng, respectively and assembled with mut-F and mut-R primers. The resulting overall product was T-cloned into Takara pMD-19 T-vector (produced by Takara BioTechnology (DALIAN)CO.,LTD.) and sequenced to confirm the sequence. The L73W and A164V mutant BL21 (DE3) dnaQ was named after mutD. | ||
[[File:Note0708.png|300px|thumb|left|Figure.1 dnaQ PCR amplification from E. Coli BL21(DE3)]] | [[File:Note0708.png|300px|thumb|left|Figure.1 dnaQ PCR amplification from E. Coli BL21(DE3)]] | ||
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== '''Week4''' == | == '''Week4''' == | ||
Revision as of 10:42, 23 September 2013
Notice:Except for specially mentioned, all the following vectors were constructed by In-Fusion HD Cloning Kit (produced by Clontech). To be brief, every fragment of desired DNA was linearized by primers designed following the protocol provided by Takara infusion cloning guide to ensure accurately 15 base pair (bp) overlap homology with each adjacent fragments. The gel-purified fragments were mixed with 2uL infusion cloning kit Premix solution and ddH2O was used to adjust the mixture to 10uL. The amount of the added fragments was based on the Takara infusion cloning guide.
Contents |
Week1(6.23-6.30)
Literature was reviewed carefully and the corresponding gene candidates were confirmed. Some of them were submitted to be synthesized. Some of students were trained for basic molecule biology experiment operation.
Week2(7.1-7.7)
The primers needed in our project is designed and synthesized. Some chemicals were also purchased. Some of students were trained for basic molecule biology experiment operation and further some characterization experiment operation.
Week3(7.8-7.14)
We cloned dnaQ gene from E. Coli BL21(DE3) by PCR and utilized overlap extension PCR to introduce mutagenesis in dnaQ by the following protocol.
Firstly, we used E. Coli BL21 (DE3) strain as template to amplify dnaQ gene by PCR with mut-F and mut-R primers, respectively. After purification of PCR product (the 〖E.Z.N.A.〗^TM Gel Extraction Kit, produced by Omega Bio-tek), it was used as template to introduce point mutation by two parallel PCRs with the following two sets of primers: mut-L73W-F and mut-L73W-R (generating L73W); mut-A164V-F and mut-A164V-R (generating A164V). The three PCR products were gel-purified and combined by 5ng, respectively and assembled with mut-F and mut-R primers. The resulting overall product was T-cloned into Takara pMD-19 T-vector (produced by Takara BioTechnology (DALIAN)CO.,LTD.) and sequenced to confirm the sequence. The L73W and A164V mutant BL21 (DE3) dnaQ was named after mutD.
Week4
Week5
Week6
Week7
Week8
Week9
Week10
