Difference between revisions of "Maeda Lab:Protocols"

Resources

<Clones>

• Aminotransferase genes from E. coli, Arabidopsis, and sorghum were synthesized through the DOE JGI Community Science Program (CSP award 503757) and are available to the scientific community.
  • If you are interested in obtaining some of these clones, please look at our DOE project website and contact Hiroshi Maeda, University of Wisconsin-Madison at maeda2@wisc.edu.

<Codes>

• Codes that we used in our studies are made available at Github for Hiroshi Maeda Lab.
  • R script for evaluating Michaelis-Menten kinetic parameters
  • ANOVA and Tukey-HSD post-hoc tests
  • Building gene trees
  • Evolutionary-rate analysis
  • RNAseq data processing
  • GO-term-enrichment

Protocols

<Molecular Biology>

• qPCR setup and analysis with the Stratagene Mx3000P
• DNA-free RNA isolation
• Arabidopsis gDNA isolation for Genotyping
• Miniprep, Alkaline Lysis
• qPCR analysis - Agilent Stratagene Mx3000
• RNA isolation from plant tissues
• E. coli competent cell preparation
• Cloning using In-Fusion system (Clontech)
• Site-directed mutagenesis using In-Fusion system

• Sequence analysis
  • 1000 Plants (1KP) BLAST
  • NCBI Blast
  • Fold & Function Assignment System (FFAS)
  • Multiple Alignment
  • BoxShade
    • Select "RTF_new" and "ALN"
  • PROMALS3D, structure-guided phylogenetic analysis
    • Choose fasta output format in PROMALS3D
    • Select all the text and save as a .txt file.
    • In MEGA go to File and select "convert file format to mega file".
    • Select the .txt file and specify fasta format. A new window will pop up to save as a new mega file.
    • Close out of this
    • Go to File, "open file/session", open the newly created mega file.
    • Run phylogenetic analyses.
  • Reverse Complement
  • Convert Sequence Format
  • Translate
  • Protein Targeting Prediction (ChloroP, Predotar,TargetP, PSORT)
  • SUBA3 (The SUBcellular localisation database for Arabidopsis proteins)
  • PhylomeDB (catalogs of gene phylogenies (phylomes)
  • Bio.Cyc collection of 7667 Pathway/Genome Databases (PGDBs), plus software tools
  • PlaNet visualization and analysis of plant co-function networks

<Biochemistry>

• Protein Modeling using Swiss-Model and PyMOL

<Analytical Chemistry>

• Tocopherol analysis - HPLC
• Amino acid analysis - HPLC and GCMS

<Plant Growth>

• Seed Sterilization - Arabidopsis, Beets

Useful links

• Research
  • KNApSAcK metabolite-species search
  • ApE plasmid Editor
  • Arabidopsis growth ARBC
  • Sorghum genome blast
  • Clint Chapple Lab at Purdue University
  • Plasmid Isolation - Mini Prep video
  • Noble Foundation Medicago insertional mutant collection
• Others
  • Waste Pickup Request
  • UW Madison Chemical Safety Dept (waste disposal etc)
  • Botany Department IT resources and introduction
  • Lab Notebook Guidelines from WARF
  • Reference managers: Mendeley and Zotero
  • How to place an Article and Book request at UW Library
  • How to merge PDF files

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