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day 1 1. Start 75 mL overnight cultures of agro (strain GV3101 C58C1 Rifr pMP90 Gmr Koncz & Schell) in YEP in 250 mL baffle flasks. 2. Grow at 28 °C shaking. 3. Prepare: ~ 3.5 L sterile H2O chilled to ...

This protocol is adapted from protocols by Nicole Bechtold (Bechtold et al. 1993) Andrew Bent (Bent et al. 1994) and Takashi Araki. No claims are made that any of the steps are necessary or ideal; th ...

1. Immerse Arabidopsis seeds in 10% Household Bleach for 20 min. 2. Rinse the seeds twice with 500 ml of sterile ddH2O and allow them to dry overnight in a laminar flow hood. 3. Push the resulting cru ...

Using this procedure it is possible to follow the development of pollen mother cells through to mature pollen. Sterile males were made using irradiation mutagenesis of Landsberg erecta seed. Buds harv ...

Materials: fixative dependent on antigen (common: 4% formaldehyde or 100% MeOH) post-fixative dependent on antigen (usually MeOH) PBS 1X PBS PBST PBS + detergent (0.05 to 0.5 % Tween or Triton) DAPI ( ...

Preparation of the probe. Probes are prepared as Digoxigenin labelled RNA . The labelling mix as well as all antibodies are purchased from Boehringer All conditions and solutions should be totally RN ...

There are two kinds of contaminants on worm plates:1. Fungi: these contaminants can come from the plates or bacteria so it is best to leave plates out after seeding for a couple of days to make sure n ...

Stockkeeping1. MechanicsMost stocks can be successfully cultured by periodic mass transfer of adults to fresh food. Bottles or vials are tapped on the pounding pad to shake flies away from the plug th ...

Equipment flies frozen in 250 ml centrifuge bottlessieves: 106 355 600 and 850 mmpaintbrushcollection tubesfunneldry icejacket hat and gloves! Preparation 1. Freeze flies in 250 ml centrifuge bottle(s ...

Gloor et al. 1993 Genetics 135:81-95We have developed a simple method for the rapid and reproducible isolation of DNA from single flies for amplification by the polymerase chain reaction (PCR) (Saiki ...

.1 RNA Probe Preparation (see Note 1) 1. 1.5 mL microcentrifuge tubes or standard 96-well V-bottom microplates.2. RNAse free water.3. T7 T3 or SP6 RNA Polymerase (Fermentas Life Sciences Burling ...

ProcedureTransfer 1.5 ml of liquid culture of yeast grown for 20 - 24 h at 30°C in YPD (1% yeast extract 2% peptone 2% dextrose) into a microcentrifuge tube. Pellet cells by centrifugation at 20000 × ...

1.Inoculate a 5 ml culture with a single colony from a YAC-containing strain of yeast and grow until saturated. Determine the number of yeast cells per milliliter. The cell count should be roughly 1 x ...

Purpose: To perform restriction digests of YACs for mapping using rare cutting enzymes or more conventional restriction endonuclease digestion. Time required: 2-4 hours Special Reagents: dependent upo ...

Pick colonies into 0.5ml of SD-Leu (or other appropriate SD medium) Vortex for 1min Leave to grow O/N for 18-24h at 30°C 230-250rpm (best in 5ml bijou) Spin yeast culture at 13000rpm for 5 min (microf ...

Grow a 5 ml YPD O/N culture inoculated with a single yeast colony at 30 deg. Transfer culture to a small 13 x 100 glass tube. Spin down cells 2 min. in tabletop centrifuge. Pour off supernatant. Wash ...

Grow 100 ml yeast cells in desired media overnight to an A600 of ~1.0 (0.6 to 1.2 works well). For growth in minimal media 1 ml of a saturated overnight culture in minimal media (Synthetic dextrose (S ...

Yeast Media: Note: Synthetic complete medium can be prepared by adding media supplements (see below). Medium using 6.7 g yeast nitrogen base without amino acids (Difco # 0919-15) can also be prepared ...

Cells are grown for 2-3 days as 1.5ml prep. under selection for the plasmid of interest. Spin cells down 2.6K for 5min. Resuspend in 1ml 0.25m NaOH/1% 2-mercaptoethanol Incubate 10min on ice. Add 0.16 ...

-Use sterile technique and sterile solutions in steps 1 to 3.- 1. Using a saturated starter culture inoculate 25 to 30 ml of appropriate media in a 125 ml flask. ***Since it is often difficult to esti ...