Care of the large fly population.

• Schedule for taking care of large population.
• Preparing grape plates for feeding adults and collecting embryos.
• Preparing cornmeal tubs for large fly population.
• Inoculating cornmeal tubs with eggs.
• Setting up and taking down large adult populations.
• Feeding large adult populations.

• Fly food for vials and bottles - updated, preferred recipe
  
• Fly food for vials and bottles - old recipe without propionic acid.
• apple juice plates

• Sources for fly supplies

• Pulling microinjection needles.
• Preparation of DNA injection mix.
• Preparing injection stocks for collection of embryos.
• Injecting embryos and identifying transformants.
• Injecting with the agar block method.
• What to do once you have a transformant?
• Southern Blot analysis to characterize transformants.
• Small scale DNA preparation from adult flies.
• Single fly DNA prep from Engels' lab for PCR analysis.
  

• "Homozygosing" and chromosome mapping.
• Mobilizing an insert to generate additional transformed lines.
• Generating homozygous flies for two genes situated on the 2^nd and 3^rd chromosomes.
• Construction of UAS-tTA derivatives.
• An excellent discussion of P-elements by Bill Engels. (Highly recommended for those of you beginning work with P-elements)

• Overview of manipulations involved in making transgenic promoter constructs (plasmid illustrations included).

• Stocks from other labs.
• Our transformants.
• Gilmour's fly rack A
• Gilmour's fly rack B
• Gilmour's fly rack C
• Gilmour's fly rack D
• Avery labels for many of the lab stocks (posted 2/18/00)

• Qualitative analysis of ß-galactosidase activity in dissected tissues.
• CPRG Assays on Crude Extract of Adult Flies
• CPRG Assays on fly larval tissues.
  
• RT-PCR analysis for salivary gland RNA
• RT-PCR analysis of Schneider 2 RNA
  

• Immunofluorescence analysis of polytene chromosomes.
• Immunofluorescence of polytene chromosomes blocked with milk.
  
• In situ hybridization for localizing sequences on polytene chromosomes.
• Permanganate genomic footprinting salivary glands (also works on fat bodies).
• Isolating DNA from glands for use in naked DNA controls.
• LM-PCR worksheet.
• LM-PCR stock solutions.
• LM-PCR primer sets.
• LM-PCR primer design
• Permanganate genomic footprinting adherant Drosophila cells.
  
• DNase I genomic footprinting salivary glands permeabilized with lysolecithin. Note, this procedure has not been reliable.
• DNase I genomic footprinting of salivary glands permeabilized with NP40.
• Chromatin Immunoprecipitation method I (our preferred method involving urea denaturation and dialysis)
• Chromatin Immunoprecipitation method II (based on the Upstate Biotechnology protocol)
  

• Biggin nuclear extract.
• Elgin nuclear extract.
• Preparation of crude TFIID, GAGA and RNA polymerase fractions from the Elgin nuclear extract.
• Promoter-independent assay for Pol II.
  
• Recombinant GAGA factor preparation.
• TCA precipitation of protein.
• Silver staining SDS-PAGE.
• A simple, sensitive and reliable silver staining procedure that is suitable for Mass Spec.
  
• SDS PAGE
• Antibody specificity test
• Screen for GST fusion proteins expressed in bacteria.
• Affinity purification of antibodies using western blotted protein antigen.
  

• Exonuclease III protection assay.
• DNase 1 footprinting proteins in solution.
• Binding mixtures of fragments to immobilized TFIID.

• In vitro Potassium Permanganate - Primer extension assay.
• In vitro transcription - Reverse transcriptase primer extension assay.

• Spermine precipitation of DNA.
• GA markers for sequencing gels.
• Alkaline plasmid mini preparation.
• Alkaline plasmid maxi preparation.
• Alkaline plasmid prep with PEG precipitation - yields DNA suitable for sequencing.
  
• Isolating DNA fragments from acrylamide gels.
• Isolating DNA fragments from agarose by centrifugation.
  
• Isolating DNA fragments from low melting point agarose.
• Isolating DNA fragment from agarose with DEAE membrane.
• Isolating DNA fragment from agarose by spinning the DNA out of the gel slice.
  
• Preparing competent E. coli using the RF1/RF2 solutions.
• Preparing competent E. coli using the PEG/DMSO solution (so-called TSS)
• Cloning PCR fragments.
• PCR screen for transformants.
  
• Inserting dephosphorylated oligos.
• Preparation of bacteria plates.
• Notes on sequencing DNA at the PSU Nucleic Acids Facility.
• End-labelling DNA with kinase.
• Useful URLs for analyzing sequences while designing cloning strategies and PCR reactions.
• Proteinase K stock
  

• EDTA titration curve
  

• Adjusting the Zeiss.
• Taking photographs with the Zeiss.

• Storage box template (for use in cataloging contents of 7 X 7 boxes)
• Storage box template (for use in cataloging contents of 9 X 9 boxes)
• Storage box template (for use in cataloging contents of 10 X 10 boxes)
• Box: Oligo A
• Box: Oligo B
• Box: Oligo C
• Box: Oligo D
• Box: Oligo E
• Box: Oligo F
  
• Other primers located somewhere in the lab.
• Box: Plasmids DSG-Z
• Box: Plasmids DSG-Y (old clones from graduate school)
• Box: Plasmids DSG 4A
• Box: Plasmids DSG 4B
• Box: Plasmids A-1
• Box: A3/Baiyong #2
• Box: plasmids DJT (Deb Taxman)
• Box: Chris' box
• Box: Tat-SF1 cloning
• Box: Boxes in the enzyme freezer
• Box: Contents of -80° freezer