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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
![](https://static.igem.org/mediawiki/2021/1/12/T--Moscow_City--tild3364-6463-4465-b335-376463323062_untitled_1.png
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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
![](https://static.igem.org/mediawiki/2021/2/23/T--Moscow_City--tild3261-6336-4137-b866-636662303361_untitled_2.png)
Cell cultures check.
Repeated heat-shock transformation of produced plasmids into chemically competent E.coli TOP10 cells with chloramphenicol (16h 37*C).
Repeated heat-shock transformation of produced plasmids into chemically competent E.coli TOP10 cells with chloramphenicol (16h 37*C).
06.10
![](https://static.igem.org/mediawiki/2021/4/43/T--Moscow_City--tild3937-3436-4465-b861-336366363934_untitled.png)
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).
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
![](https://static.igem.org/mediawiki/2021/2/2b/T--Moscow_City--tild3032-6666-4961-b963-303531653539_-1.png
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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-).
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
![](https://static.igem.org/mediawiki/2021/c/c5/T--Moscow_City--tild3664-3765-4239-a334-363164343636_untitled_3.png
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Screening of prepared mixes on gel electrophoresis. Only dcas13a-C_lact for inteine chimeric protein was successful
11.09
![](https://static.igem.org/mediawiki/2021/c/c4/T--Moscow_City--tild3934-6332-4636-b565-393062373839_untitled_4.png)
Cell cultures check: all seems well. Rare colonies.
Eurogene Cleanup Standart:
https://evrogen.ru/kit-user-manuals/BC022.pdf
GG:
https://www.neb.com/-/media/nebus/files/manuals/manuale1601.pdf
Qubit concentration measurement:
The Qubit dsDNA HS (High Sensitivity) Assay
PCR Q5-HF:
https://international.neb.com/protocols/2012/12/07/protocol-for-q5-high-fidelity-2x-master-mix-m0492
Next steps: