Protocols from Prashant (2016-2024)

These are all my protocols developed in Shamoo, Stadler and Silberg labs over the years. I am uploading them to the stadler github for easier accesibility than my original OneNote. Contact me if you want to access more experimental details etc. for access to my original lab notebook.

Outline of protocols

• Protocols
  • 1-step RT-PCR NEB LunaScript (for long amplicons)
  • 2xYT broth
    • LBMC broth (S meliloti)
    • YT-Ca broth (S meliloti)
  • Agar media
    • HPC agar
    • LB agar - quick
    • R2A agar
  • Column purification (Zymo)
    • Exo-CIP rapid enzymatic cleanup kit
    • Magnetic bead DNA size selection - Solid phase reversible immobilization (AmpureXL etc.)
  • Competent cells
    • ** transformation**
      • Competent cells transformation
      • E. coli ccmb80
      • E. coli Electrocompetent
      • E. coli TSS competent
      • Electrocomp (V. natriegensVmax)
      • Heat shock Vmax cold, like CCMB80Inoue
      • Heat shock Vmax Room temp (like Hanahan)
      • Pseudomonas electrocompetent-frozen
      • Zymo - mix and go
  • Culturing in blocks has lot more cell density than tubes
  • Direct qPCR protocol
    • Direct qPCR buffer
    • Direct qPCR literature
    • Direct RT-qPCR RNAgem kit
    • IGEM (dry-heating)
  • DNA decontamination resources
  • E. coli ccmb80
    • CCMB80 buffer
  • Electrocomp (V. natriegens
    • Vmax)
      • Vmax electrocomp tabular
  • Flow cytometry (Guava)
    • fcs tutorial and terminology
    • Guidelines - Sample processing etc
    • Sony FACS cell sorter in Keck hall
    • Sony flow cyt SEABRC
    • Washing cells with PBS+Tween
  • Gel extraction protocol (ideal)
    • Gel quantification with ImageJ
    • Zymo gel purification
  • Genewiz sequencing guidelines
    • Ez-amplicon sequencing notes
  • GS-MS manual
  • Media
    • 2xYT broth
    • 10x V2 salts
    • Endo agar (coliform)
    • LB media- Lysogeny broth, Miller
    • LBmc other bacterial media
    • R2A agar
    • Selective yeast medium
    • SOB
    • SOC - recovery media
    • Terrific Broth
    • TSS media
    • Universal media (IGEM 2018)
    • YPD (Yeast)
  • Microbiology
    • Antibiotics - other organisms IGEM 2018
    • Bacillus resources
    • broad host origins list
    • General conjugation protocol
    • Natural transformation Acinetobacter Baylyi ADP1
    • Pseudomonas resources
    • Vmax (Vibrio natrigens) resources
    • Yeast resources
  • Minimal media
    • M9cas
  • Molecular biology
    • Colony PCR
    • Column purification (Zymo)
    • Gel extraction protocol (ideal)
    • Genewiz sequencing guidelines
    • Genomic DNA extraction
    • Gibson assembly homemade (ITA)
    • LAB buffer - Gels
    • Lambda red recombineering protocol
    • List of Golden gate enzymes
    • Nanopore sequencing
    • Phusion DNA Polymerase protocol 2kb
    • Plasmidsaurus - whole plasmid nanopore service
    • Qiagen miniprep
    • Restriction buffers NEB composition
    • RNA extraction Maxwellmagnetic
    • Shyam's protocols
  • Nanopore sequencing
    • Analysis pipelines Plasmidsaurus WGS
    • Rapid barcoding kit (SQK-RBK004)
    • Transition away from R9 chemistry
  • Qiagen miniprep
    • Mira prep
  • qPCR protocols Dye based
  • Replica plating
  • RNA extraction Maxwell
    • magnetic
      • DNAse treatment of RNA extracts
  • Shyam's protocols
    • BTK - details broad
    • BTK frequently used parts
    • BTK notes of wisdom
    • BTK overview figures
    • Terminators to couple - guidelines
  • Solubility of chemicals
  • Vmax (Vibrio natrigens) resources
    • Vibrio antibiotic selection
    • Vibrio compatible plasmids
    • Vibrio media, growth rates

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