Human codon-optimized base editing constructs were a kind gift from David Liu; pCMV_ABEmax_P2A_GFP (plasmid #112101; Addgene), pCMV_AncBE4max_P2A_GFP (plasmid#112100; Addgene). pCMV_SpCas9-NG_ABEmax_P2A_GFP, pCMV_SpCas9-NG_AncBE4max_P2A_GFP and pCMV_SaKKH_AncBE4max_P2A_GFP were constructed by PCR amplification (Q5, NEB) amplifying everything except for SpCas9 using pCMV_ABEmax_P2A_GFP and pCMV_AncBE4max_P2A_GFP. Coding sequences for SpCas9-NG and SaKKH were PCR amplified using the following plasmids NG-ABEmax (plasmid #124163; Addgene) and SaKKH-ABEmax (Plasmid #119815; Addgene) that were a kind gift from David Liu. Coding sequences and plasmid backbones were combined using the NEBbuilder HiFi DNA assembly mastermix (NEB) and subsequently transformed using OneShot Mach1t1 (Thermo Fisher Scientific) cells and plasmid identity was checked by Sanger sequencing (Macrogen). The empty sgRNA plasmid backbone for SpCas9 and its derivatives was a kind gift from Keith Joung (BPK1520, Addgene plasmid #65777). Spacer sequences targeting all genes in this study were cloned in the sgRNA plasmid backbone using inverse PCR (Q5 NEB) and subsequently transformed using OneShot Mach1t1 (Thermo Fisher Scientific) cells and plasmid identity was checked by Sanger sequencing (Macrogen). Primer sequences for sgRNA generation can be found in Supplementary Table 3 .
One shot mach1 t1
Manufactured by Thermo Fisher Scientific
The One-Shot Mach1 T1 is a laboratory instrument designed for high-throughput DNA transformation. It facilitates the introduction of genetic material into competent bacterial cells through an efficient electroporation process. The device streamlines the transformation workflow, allowing for rapid and consistent results.
Automatically generated - may contain errors
Lab products found in correlation
Hifi dna assembly master mix, by New England Biolabs (1 mentions)
Sanger sequencing, by Thermo Fisher Scientific (1 mentions)
Ng abemax, by Addgene (1 mentions)
Pcmv abemax p2a gfp, by Addgene (1 mentions)
Pcmv ancbe4max p2a gfp, by Addgene (1 mentions)
Bpk1520, by Addgene (1 mentions)
Abi 3730xl dna analyzer, by Thermo Fisher Scientific (1 mentions)
7 protocols using one shot mach1 t1
1
Codon-Optimized Base Editing Constructs
2
Yeast Display of SrtAβ Mutants
3
Identifying Aptamers from SELEX Cloning
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The individual aptamers present at the last round of DNA or RNA SELEX processes were cloned. With that aim, the products of the PCR-amplified DNA (or RT-PCR-amplified RNA) were cloned using the TOPO TA cloning PCR II Kit (ThermoFisher) and the recombinant plasmids were used to transform One Shot Mach1-T1 (ThermoFisher) competent E. coli strain. Then, 26 DNA and 32 RNA individual aptamers were sequenced by capillary electrophoresis using an ABI 3730xl DNA Analyzer (ThermoFisher) and following standard protocols.
4
Site-Directed Mutagenesis of tcaA Gene
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Site-directed mutagenesis was used to mutate the wild type tcaA gene (Phe290) to the SNP tcaA (Ser290). Primers (5’ ttttcaagttTcaaaacgtatggtc 3’, 5’ atacgttttgaAacttgaaaatgcc 3’) were designed to have a complementary overlap of 21 nucleotides at the 5’ end, with the nucleotide to be mutated at the centre of the overlap (the primers had non-complementary regions at the 3’ end to facilitate annealing on the template). The ptcaA template was diluted to 33 ng/µl, and 1 µl was amplified using these primers and Phusion DNA polymerase. The PCR product was subsequently checked by agarose gel electrophoresis and was treated with 1 µl of DpnI directly in the PCR mix for 1 hr at 37°C. Then, 1 µl of the mixture was used to transform One Shot Mach1 T1 (Thermo Fisher) competent cells by heat shock then into RN4220 and finally tcaA::tn by electroporation.
5
Yeast Display of SrtAβ Mutants
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
SrtAβ was subcloned into pET29 and used as PCR template for reactions with primers in Supplementary Table 3 . Following USER assembly or KLD ligation, products were transformed into Thermo Fisher One-Shot Mach1 T1 Chemically Competent cells. Following sequence verification, the reversion mutants were amplified out of the pET29 backbone with HR primers and transformed into ICY200 with NheI/BamHI-digested pCTCon2CTev vectors in a 5:1 insert:backbone mass ratio to yield yeast bearing single reversion mutants for flow cytometry analysis.
6
Site-Directed Mutagenesis of tcaA Gene
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Site-directed mutagenesis was used to mutate the wild type tcaA gene (Phe290) to the SNP tcaA (Ser290). Primers (5’ ttttcaagttTcaaaacgtatggtc 3’, 5’ atacgttttgaAacttgaaaatgcc 3’) were designed to have a complementary overlap of 21 nucleotides at the 5’ end, with the nucleotide to be mutated at the centre of the overlap (the primers had non-complementary regions at the 3’ end to facilitate annealing on the template). The ptcaA template was diluted to 33 ng/µl, and 1 µl was amplified using these primers and Phusion DNA polymerase. The PCR product was subsequently checked by agarose gel electrophoresis and was treated with 1 µl of DpnI directly in the PCR mix for 1 hr at 37°C. Then, 1 µl of the mixture was used to transform One Shot Mach1 T1 (Thermo Fisher) competent cells by heat shock then into RN4220 and finally tcaA::tn by electroporation.
7
Site-directed Mutagenesis of tcaA Gene
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Site directed mutagenesis was used to mutate the WT tcaA gene (Phe290) to the SNP tcaA (Ser290). Primers (5” ttttcaagttTcaaaacgtatggtc 3”, 5” atacgttttgaAacttgaaaatgcc 3”) were designed to have a complementary overlap of 21 nucleotides at the 5’ end, with the nucleotide to be mutated at the centre of the overlap (the primers had non complementary regions at the 3’ end to facilitate annealing on the template). The ptcaA template was diluted to 33 ng/μl, and 1ul was amplified using these primers and Phusion DNA polymerase. The PCR product was subsequently checked by agarose gel electrophoresis and was treated with 1 μl of DpnI directly in the PCR mix for 1 h at 37°C. Then 1 μl of the mixture was used to transform One Shot™ Mach1™ T1 (ThermoFisher) competent cells by heat shock then into RN4220 and finally tcaA::tn by electroporation.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!