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E2621

Manufactured by New England Biolabs
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The E2621 is a laboratory instrument produced by New England Biolabs. It is designed for nucleic acid amplification and detection, a core function in molecular biology research and diagnostics.

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Lab products found in correlation

Phusion high fidelity dna polymerase, by New England Biolabs (2 mentions)

3 protocols using e2621

1

SARS-CoV-2 Spike Protein Mutant Generation

Cited 2 times
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The codon-optimized SARS-CoV-2 spike (Wuhan) in pcDNA3.1 was a gift from P. McKay [46 (link)], Imperial College London. Site directed mutagenesis using the QuikChange Lightning Site-Directed Mutagenesis Kit (210518) was used to introduce the spike mutations of Alpha. The BA.1 spike plasmids were a kind gift from T.Peacock. To generate the mutant spike plasmids and domain swap chimeric spike plasmids a combination of site directed mutagenesis and DNA assembly was used (NEB #E2621).
The C-terminal end of the Alpha spike sequence was tagged with mGreenLantern [27 (link)] subcloned from pcDNA3.1-mGreenLantern, which was a gift from Gregory Petsko (Addgene plasmid # 161912; http://n2t.net/addgene:161912; RRID:Addgene_161912) using a glycine-serine rich linker (GSGGSGSGG) [47 (link)] to replace the last 21 amino acids of the spike sequence.
pmScarlet_C1 [28 (link)] was a gift from Dorus Gadella (Addgene plasmid # 85042; http://n2t.net/addgene:85042; RRID:Addgene_85042)
pcDNA3-sACE2(WT)-Fc(IgG1) [47 (link)] was a gift from Erik Procko (Addgene plasmid # 145163; http://n2t.net/addgene:145163; RRID:Addgene_145163)
mScarlet was subcloned from pmScarlet_C1 and tagged to the end of the Fc tag of sACE2(WT)-Fc(IgG1) to create sACE2(WT)-Fc(IgG1)-mScarlet.
Kugathasan R., Sukhova K., Moshe M., Kellam P, & Barclay W. (2023). Deep mutagenesis scanning using whole trimeric SARS-CoV-2 spike highlights the importance of NTD-RBD interactions in determining spike phenotype. PLOS Pathogens, 19(8), e1011545.
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2

eRNA Cloning and Mutant Construction

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eRNA sequences, derived from hg38 assembly, were cloned from genomic DNA extracted from MCF-7 cells (Qiagen, 51304) using the four restriction sites between the sgRNA scaffold and the polyA signal. eRNAs were amplified using Phusion High-Fidelity DNA polymerase according to the manufacturer’s protocol (NEB, M0530L). The reaction was then separated on an agarose gel, and PCR product corresponding to the correct size was purified (Qiagen, 28704), then ligated into digested pInd20 (with or without sgRNA), transformed, selected, purified, and verified as above.
For PRRX2 eRNA MT and WT.AS constructs, a reverse primer encompassing the mutant or antisense sequence was used in combination with the PRRX2 eRNA forward primer. pInd20 digested with the appropriate enzymes (BclI/SbfI) was used for Gibson cloning (NEB, E2621), transformed, selected, purified, and verified as above.
For PRRX2 ΔFERM, a gBlock (IDT) containing the FERM-deletion eRNA was ordered, amplified, and cloned into pInd20. For UBE2E2 ΔFERM, two overlapping fragments without the FERM were PCR amplified and assembled using Gibson assembly. All plasmids were transformed, selected, purified, and verified as above.
Hou T.Y, & Kraus W.L. (2022). Analysis of estrogen-regulated enhancer RNAs identifies a functional motif required for enhancer assembly and gene expression. Cell reports, 39(11), 110944.
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3

eRNA Cloning and Mutant Construction

Check if the same lab product or an alternative is used in the 5 most similar protocols
eRNA sequences, derived from hg38 assembly, were cloned from genomic DNA extracted from MCF-7 cells (Qiagen, 51304) using the four restriction sites between the sgRNA scaffold and the polyA signal. eRNAs were amplified using Phusion High-Fidelity DNA polymerase according to the manufacturer’s protocol (NEB, M0530L). The reaction was then separated on an agarose gel, and PCR product corresponding to the correct size was purified (Qiagen, 28704), then ligated into digested pInd20 (with or without sgRNA), transformed, selected, purified, and verified as above.
For PRRX2 eRNA MT and WT.AS constructs, a reverse primer encompassing the mutant or antisense sequence was used in combination with the PRRX2 eRNA forward primer. pInd20 digested with the appropriate enzymes (BclI/SbfI) was used for Gibson cloning (NEB, E2621), transformed, selected, purified, and verified as above.
For PRRX2 ΔFERM, a gBlock (IDT) containing the FERM-deletion eRNA was ordered, amplified, and cloned into pInd20. For UBE2E2 ΔFERM, two overlapping fragments without the FERM were PCR amplified and assembled using Gibson assembly. All plasmids were transformed, selected, purified, and verified as above.
Hou T.Y, & Kraus W.L. (2022). Analysis of estrogen-regulated enhancer RNAs identifies a functional motif required for enhancer assembly and gene expression. Cell reports, 39(11), 110944.
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