实验动物模型

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 ( ...

I. MEDIUMA. S2 S2C Sundefined S2R+ S3 Kc(167) DL1 & DL2 cells: Schneider''s/ 10% FBS/ PS450 ml Schneider''s medium (Gibco #11720-034)(pour off 50 ml into Falcon to save as serum-free~undefined*50_ml_Fetal_Bovine_Se_...

.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 ...

Purpose: To isolate intact high molecular weight DNA from yeast cells for subcloning and rare cutting restriction enzyme analysis. One can expect a yield of 100-200 � of DNA per prep. Time required: 6 ...

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 ...

Grow 10ml YPD cultures o/n. Figure out cell density; inoculate 30 ml YPD and grow o/n so that cell density is approximately 2 x 108 cells/ml the next morning.Spin down cells in 50 ml sterile conical t ...

YEPD (non-selection)-1% yeast extract-2% peptone-1.5% agar (if needed for plates)After autoclaving add glucose to 2% by adding 100 ml of 20% solution to each litre media.SYNTHETIC MEDIA (selective med ...

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 ...

Procedure pick one colony inoculate in 3 ml of the appropriate media grow at 30° overnight pellet the cells (5 min 5000g) wash 1X in sterile ddH2O re- suspend the pellet in 200 µl EthOH (optional: +2 ...

This method gives yeast nuclei which look nearly purified microscopically. Nuclei isolated in this way do not give active transcription extracts when the nuclei are salt extracted and the resulting pr ...

This is the preferred method for yeast RNA preparation use Gloves and RNAse free solutions throughout.1. Use a YPD overnight culture to innoculate fresh YPD media and grow cells at 30 degrees overnigh ...

AbstractWhen more than one bait will be used to screen a single library significant time and resources can be saved by performing the interactor hunt by interaction mating. In this protocol one strain ...

Express fusion proteins as per the GST-fused protocol up to Step 7 (Day 3). All steps in protein purification should be done at 4° C unless otherwise stated.Pre-wash 10-20 ml of New England Biolabs am ...

Protocol: Fast yeast transformationAdd 50 µl carrier DNA to a 1.5 ml tube. scrap cells from plate and add to the carrier DNA. Add in the following order: 1-2 µl DNA-plasmid (up to 30-50 µl) 240 µl PEG ...

Day 0 Make sure you have the necessary solutions (instructions for how to make them can be found here): Single-stranded carrier DNA PEG 3350 50% w/vol 1.0 M LiAc Make sure you have enough plasmid DNA ...

Short term storageYeast cultures are stable for 1-2 weeks when refrigerated. Petri dishes should be sealed or in plastic bags. Medium term storageYeast can be preserved for up to a year on YPD agar me ...

Grow cells Harvest Spin down Wash with PBS Spin down enough cells to collect a 50-100 µL cell pellet in a FastPrep tube Prepare lysis buffer (beforehand) and chill on ice PBSMT: 2.5 mM MgCl2 3 mM KCl ...

Pad Preparation1. Microwave 2% agarose (mix of low-melt and normal to taste) in Thorn media (see below). (If you have used different percentages of agarose pads please let us know bruno) 2. Apply 2mL ...

OverviewBlotting for large V5-tagged proteins in S. cerevisiae MaterialsY-PER (Pierce) Halt EDTA-free Protease Inhibitor (Pierce) NuPAGE Novex Bis-Tris 4-12% mini gels (Invitrogen NP0321BOX) Prestaine ...

MaterialsYPD plates YPD 1 M sorbitol 182 g/l (Sigma S7547) 2 M sorbitol 36.4 g/100 ml SCE (per liter) 1 M sorbitol (182 g) 100 mM citric acid trisodium salt dihydrate (29.4 g) 10 mM EDTA 20 ml of 500 ...