A backbone containing two inverted loxP sites3 (link) was used to clone several empty reporter vectors containing a 60-bp universal entry site with a central EcoRV restriction site, followed by a promoter (pCAGGS, hPGK and Ef1alpha) that drives an eGFP or mScarlet gene for the ChroMM and EpiTF screens, respectively, followed by a downstream BGH-poly(A) and a WPRE sequence. Individual ICR or control sequences were amplified from genomic DNA (Supplementary Table 1). Gibson assembly was performed according to the NEB Gibson Assembly Master Mix protocol. In vitro methylation was performed with up to 40 μg plasmid DNA using the NEB M.SssI methyltransferase in two consecutive reactions of at least 4 h with 600 μM SAM (NEB, B9003S) and 1.5 U M.SssI (NEB, M0226L) per microgram DNA. Complete methylation of plasmids was confirmed by using the CpG methylation sensitive restriction enzyme HpaII (NEB) and a methylation insensitive control reaction with MspI (NEB). Cell lines were generated as described before. Individual clones were genotyped using PCR with primers spanning the loxP sites. Methylation of the integrated reporter construct was validated on selected clones.
Gibson assembly master mix protocol
Manufactured by New England Biolabs
The Gibson Assembly Master Mix protocol is a molecular cloning technique used for the seamless assembly of multiple DNA fragments. The protocol involves the use of a proprietary enzyme mix that enables the efficient joining of DNA sequences with overlapping regions. This procedure facilitates the construction of recombinant DNA molecules without the need for restriction enzymes or ligation steps.
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Lab products found in correlation
2 protocols using gibson assembly master mix protocol
1
Methylation-Sensitive Chromatin Reporters
2
Cloning Methylation-Sensitive Reporters
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A backbone containing two inverted loxP sites3 (link) was used to clone several empty reporter vectors containing a 60-bp universal entry site with a central EcoRV restriction site, followed by a promoter (pCAGGS, hPGK and Ef1alpha) that drives an eGFP or mScarlet gene for the ChroMM and EpiTF screens, respectively, followed by a downstream BGH-poly(A) and a WPRE sequence. Individual ICR or control sequences were amplified from genomic DNA (Supplementary Table 1 ). Gibson assembly was performed according to the NEB Gibson Assembly Master Mix protocol. In vitro methylation was performed with up to 40 µg plasmid DNA using the NEB M.SssI methyltransferase in two consecutive reactions of at least 4 h with 600 µM SAM (NEB, B9003S) and 1.5 U M.SssI (NEB, M0226L) per microgram DNA. Complete methylation of plasmids was confirmed by using the CpG methylation sensitive restriction enzyme HpaII (NEB) and a methylation insensitive control reaction with MspI (NEB). Cell lines were generated as described before. Individual clones were genotyped using PCR with primers spanning the loxP sites. Methylation of the integrated reporter construct was validated on selected clones.
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