pET-pAG-Hia5
(Plasmid #174373)

• Purpose: Plasmid for IPTG-inducible expression and purification of protein A/G-Hia5-6xHis (pAG-Hia5) for antibody directed DNA (adenine) methylation (as in DiMeLo-seq)
• Depositing Lab: Aaron Straight
• Publication: Altemose et al Nat Methods. 2022 Jun;19(6):711-723. doi: 10.1038/s41592-022-01475-6. Epub 2022 Apr 8.

Backbone

• Vector backbone: pET
• Total vector size (bp): 6741
• Vector type: Bacterial Expression

Growth in Bacteria

• Bacterial Resistance(s): Kanamycin, 50 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Growth instructions: For protein expression, use T7 Express lysY competent E. coli cells (NEB #C3010I) See protocol: dx.doi.org/10.17504/protocols.io.bv82n9ye
• Copy number: Unknown

Gene/Insert

• Gene/Insert name: protein AG - hia5
• Insert Size (bp): 1500
• Tags / Fusion Proteins:
  • Protein A/G (N terminal on insert)
  • 6xHis (C terminal on backbone)

Resource Information

• Addgene Notes:
  • Addgene Diagnostic Digest

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
• Industry Terms:
  • Not Available to Industry

Depositor Comments

Protocol for purification and expression can be found at: dx.doi.org/10.17504/protocols.io.bv82n9ye
Application for DiMeLo-seq is described in: doi.org/10.1101/2021.07.06.451383
Protocol was adapted and modified from: doi.org/10.1126/science.aaz1646

This plasmid has been observed to occur in our stock as a plasmid dimer. Dimerization often does not impact plasmid function, but may reduce transformation and transfection efficiencies. You may need to screen multiple colonies to isolate the monomeric version of this plasmid. If you still have trouble isolating the monomeric version, you might consider linearizing, gel extracting, re-ligating, and transforming the plasmid in a recombinase-deficient strain.