Team:Moscow City/Lab book

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Lab book
10.09
Primers delivered. Dilution of new primers to 10 μL/mL. We didn`t phosphorylate our primers. You don't need phosphorylated primers for sticky end cloning. Use normal primers and the 5' phosphates will be there after restriction digest.
15.09
Successfull PCR of N-term beta-lactamase (N-lact), N-term intein (N-int) and unsuccessful PCR of BsmBI-dCas13a (N-dCas13a). Gel electrophoresis of N-lact and N-int, gel extraction.
16.09
PCR, gel electrophoresis and gel extraction of C-term beta-lactamase (C-lact).
17.09
PCR, gel electrophoresis and gel extraction of Bsm-dCas13a (C-dCas13a), N-dCas13a, C-lact.
23.09
PCR and gel electrophoresis of N-Cas, C-term-int (both successful) and C-Cas (unsuccessful). PCR (temperature gradient) and gel electrophoresis of C-Cas (55.4*C and 57.3*C successful).
24.09
Gel extraction of N-Cas and C-int.
27.09
Gel extraction of C-lact. PCR of N-lact (for intein+ and intein- assemblies) and C-lact. Synthesis of different primer for C-lact on Kilobaser (cause we had troubles with its ancestor)
28.09
PCR of C-lact (int-). Gel electrophoresis of N-lact (int+), N-lact (int-), C-lact (int+), C-lact (int-). All successful excluding C-lact (int-).
29.09
Gel extraction: C-lact (int+, N-lact (int+), N-lact (int-).
01.10
Golden Gate cloning into pGGAselect for N-dCas13a with N-lact and C-dCas13a with C-lact. Heat-shock transformation of produced plasmids into chemically competent E.coli TOP10 cells with chloramphenicol. Incubation of cell cultures at (16h 37*C and 52h 4*C and 37*C 3h and room temperature 24h).
04.10
Cell cultures check.
Repeated heat-shock transformation of produced plasmids into chemically competent E.coli TOP10 cells with chloramphenicol (16h 37*C).
06.10
Cell cultures check.
Golden Gate cloning into pGGAselect for:
1) N-dCas13a with N-lact (int+);
2) N-dCas13a with N-lact (int-);
3) C-dCas13a with C-lact (int+);
4) C-dCas13a with C-lact (int-).
Heat-shock transformation of produced plasmids into chemically competent E.coli TOP10 cells with chloramphenicol. Incubation of cell cultures at (room temperature 64h).
07.10
Cell cultures check.
Screening preparation of single colonies with Quick-Load® Taq 2X Master Mix:
1) N-dCas13a with N-lact (int+);
2) N-dCas13a with N-lact (int-);
3) C-dCas13a with C-lact (int+);
4) C-dCas13a with C-lact (int-).
8.10
Screening of prepared mixes on gel electrophoresis. Only dcas13a-C_lact for inteine chimeric protein was successful
11.09
Cell cultures check: all seems well. Rare colonies.