Lab Protocols

Abstract The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental pathways. Several powerful and well established transgenic technologies are available to manipulate gene expression levels in mouse, allowing for the generation of both loss- and gain-of-function models. However, the generation of mouse transgenics is both costly and time consuming. Alternative methods of gene manipulation have therefore been widely sought. In utero electroporation is a method of gene delivery into live mouse embryos 1,2 that we have successfully adapted 3,4 . It is largely based on the success of in ovo electroporation technologies that are commonly used in chick 5 . Briefly, DNA is injected into the open ventricles of the developing brain and the application of an electrical current causes the formation of transient pores in cell membranes, allowing for the uptake of DNA into the cell. In our ha

Abstract Dense array electroencephalography ( d EEG), which provides a non-invasive window for measuring brain activity and a temporal resolution unsurpassed by any other current brain imaging technology 1,2 , is being used increasingly in the study of social cognitive functioning in infants and adults. While d EEG is enabling researchers to examine brain activity patterns with unprecedented levels of sensitivity, conventional EEG recording systems continue to face certain limitations, including 1) poor spatial resolution and source localization 3,4 ,2) the physical discomfort for test subjects of enduring the individual application of numerous electrodes to the surface of the scalp, and 3) the complexity for researchers of learning to use multiple software packages to collect and process data. Here we present an overview of an established methodology that represents a significant improvement on conventional methodologies for studying EEG in infants and adults. Although se

Plastic labware is a crucial ingredient of any laboratory. So it is imperative that all the plastic labware is properly cleaned and cared for to get the right result. In this page different aspects of caring as well as cleaning of plasticware has been provided to guide the users to accurate experiment results. Effect of Chemicals on Plastic Labware Certain types of chemicals like oils, detergents, lubricants,pure water and surface additives in presence of tensile stress can result in cracking of the plastic ware. Extended exposure to strong oxidising agents are also known to cause embrittlement and failure. A Chemical affects the strength, flexibility, appearance, dimensions as well as weight of plastic labware. The intensity depends upon. Length of exposure Temperature Concentration  Effect of Heat on Plastic Labware Plastic labware should never be allowed to come in direct contact with a flame or put on a hotplate surface. 

For getting successful results from the experiments performed in a laboratory, it is essential that the lab glassware gets perfectly cleaned. However cleaning laboratory glassware is not a very easy task. Laboratory glassware cleaning has to be done physically as well as chemically so that they become free of any germs. However it is to be kept in mind that as such there is no single best laboratory glassware cleaning solution that can be termed as the "best" for all conditions. Laboratory Glassware Cleaning Basics Laboratory Glassware Cleaning If the lab glassware is cleaned immediately after use then the problems associated with dirt can be controlled to a great extent. If detergent is used for laboratory glassware washing, it should be the one that is specially designed for lab glassware cleaning. These detergents are always to be used in place of the normal dish washing detergents used in homes. It has been seen that for cleani

Fritted glassware used in the labs unless properly taken care of and used in proper conditions can result in breakage as well as inaccurate results. This section highlights some of the important things regarding the cleaning and usage of Fritted Glassware. Cleaning of Frittedware A new fritted ware like a filter is required to be washed by suction. For this hot hydrochloric acid is used and followed by rinsing with water. This treatment effectively removes loose particles like dust. Like all glassware it is also advisable to clean all Fritted ware as soon as possible after use. This helps to extend their life. For a Fritted filter most of the precipitates gets removed from the filter surface with a simple rinsing by water. However this is to be done from the reverse side with a water pressure not exceeding 15 lbs./sq. in. Alternatively, water is drawn through the filter from the reverse side using a vacuum pump is equally effec

A plastic labware that is used repeatedly for experiments needs to be free from any contaminants to ensure a precise result. To ensure this plastic labware are sterilised. In this section different techniques of sterilisation are discussed. Autoclaving : Autoclaving of plastic labware is done at 121°C, 15 psi for half an hour. Before autoclaving process ensure that the caps are properly loosened or removed so as to check any accidental implosion. It has been seen that few chemicals with no appreciable effect on plastic resins at room temperature causes deterioration during autoclaving temperatures. That is why they must be removed with distilled water prior to autoclaving. Resins like Polymethylpentene, PFA, Polypropylene, polypropylene copolymer can be autoclaved repeatedly at 121°C/15 psi with a 15 minutes cycle length. Polysulfone (PSF) and polycarbonate (PC) although autoclavable, but gets we

If you want to prolong the life of laboratory utensils made of stainless steel then it is necessary to take proper care of them. The following points given below give some useful hints in caring for stainless steel laboratory utensils. Using Proper Tools : While cleaning stainless steel products , it is better to use non-abrasive tools. Soft cloths and plastic scouring pads if used does not cause any damage to the steel’s passive layer. Using stainless steel pads is also a good option but the scrubbing motion must be directed towards the polishing marks. Cleaning Along the Polish Lines : Often some types of stainless steel lab products come with polishing lines or “grain”which are visible. If visible lines are seen then scrubbing should be done in a motion which is parallel to them. In case grains are not seen a soft cloth or plastic scouring pad can be used. Use of Appropriate Cleaners : For stai

Safety Signs are of a paramount importance in all labs. It is very essential to povide complete protection to those working in laboratories. The handling of different chamicals, acids and other substances is to be done with great care as these have some inherent features which can be harmful to health. As such all laboratories, whether in schools, universities, hospitals, health centers, test centers should promote health and safety by proper labeling of laboratory equipments, including lab safety signs and symbols. The following are some of the important signs that can be found in the science laboratories warning the users about the possible dangers. Sign for combustible material Sign for corrosive material Sign for toxic chemicals Sign for environmental hazard Sign for eyewash station

Laboratory safety equipment is a must in any laboratory especially those dealing in chemical or bio-hazard labs. These days owing to constant research, laboratory safety equipment & lab safety supply are coming with more advanced features that minimizes the risk factors. Whatever be the safety equipment available what is needed is proper validation and regular checks to keep them in operating condition so as they can be easily used when necessary. There are different laboratory safety equipments for varied purpose. Laboratories are places of researches and experiments. Here new things are discovered and invented. In the process the researchers, scientists or lab professional come across a number of equipments, apparatus, samples and chemicals. Regular dealing with all these things can pose serious health problems to those working in labs. Keeping their health in view, a number of laboratory safety measures are taken, which require different types of lab safety equipment and lab

It is very essential to follow safety rules in a laboratory, specially in a chemistry laboratory as most of the chemicals and acids used in various experiments are flammable and can be very dangerous if not properly handled. If proper safety rules are maintained, accidents and casualty can be prevented. Here are some of the basic safety rules, which should be followed while working in a chemistry lab, while handling various chemistry lab equipment: Chemistry Laboratory Safety The most important safety rule to consider wearing appropriate clothing as most accidents occur due to improper dress. The lab assistants or the researchers spill acid on themselves or others, which give a burning sensation and people actually set themselves on fire accidentally. Long and loose clothes are strictly prohibited in labs. Long hair should be properly tied. Wear clothes that are made of materials that fire proof. Lab coat and safety goggles are advised to put on during working in ch

Some of the important considerations in the process of measurement are described here. Accuracy : By Employing a suitable tool a measure of a physical quantity can be known. The reading's accuracy depends on two factors. The device as well as the person who takes the measurement. Therefore accuracy can mean the degree up to which a measured value conforms with a standard or true value. Least Count : A measuring instrument's accuracy depends upon its least count. Least count of the instrument is the least distance which it can measure accurately. Measuring instruments such as vernier callipers, micrometer, screw gauge, etc. show different levels of accuracy in comparison to each other. For example the least count of a screw gauge is 0.01mm while that of a vernier calliper is 0.1mm.  Significant Figures : Significant figures refer to those digits about which we are perfectly sure of plus those digits about which we are not that sure. However, they have a value which is meani

It is a known fact for anyone working in a laboratory be it the students, the researchers, scientists or lab assistants, it is necessary to follow safety rules and guidelines for their own safety. All laboratories have their lab safety symbols and lab safety equipments like goggles, mask, dress etc. At the same time, it is also very essential that a laboratory is well-maintained and taken care of so as to prevent any kind of dangers involved while working in a lab. As per rule, there should be routine maintenance and safety checks in a laboratory. Infact, it should be the duty of every person working in a laboratory to do a quick safety check after the completion of each experiment and before the starting of a new one. Or a responsible personnel should be assigned the duty to clean the laboratory and perform simple maintenance for lab care and safety . Laboratory Maintenance Presenting below few points which should be considered for laboratory maintenance: Bench:

Make up HCG solution of 500IU per 1 mL. (in 2500IU vial add 5cc 0.9% saline) Store HCG solution at 4C for up to three months. Day 1: Morning (9am)   – Inject male with 150IU. Place in separate tank. Day 2:               Morning (9am)   – Inject female with 250IU.   Place in breeding tank.                                        Inject male with 150IU.   Return to separate tank.               Evening (5pm) – Inject female with 500IU .   Return to breeding tank.                                          Place male into breeding tank with female. Day 3:   Collect lots of fertilized eggs.   If no eggs, re-inject female with 500IU   and re-inject male with 150IU  

Fixation of Xenopus tadpoles for cryosectioning Fix tadpoles in 4%PFA/0.1M PB for 1h at room temperature on the shaker Transfer tadpoles to cold Dent’s Fixative (80% Methanol/ 20% DMSO, store at -20°C) Fix overnight at -20°C (at this step tadpoles can be stored for a longer time at -20°C) Wash tadpoles in 100mM Tris/HCl pH 7.4, 100mM NaCl for 1h at room temperature on the shaker Transfer tadpoles to 15% Fish Gelatin/ 15% Sucrose, incubate o/n at room temperature on the shaker Transfer tadpoles to 25% Fish Gelatin/ 15% Sucrose, incubate o/n at room temperature on the shaker Embed tadpoles in 20% Fish Gelatin/ 15% Sucrose for cryosectioning Fish Gelatin, from Norland (HiPure Liquid Fish Gelatin HP-03) Attention when preparing the solutions since the fish gelatin stock solution is just a 45% solution Don’t measure fish gelatin with cylinder but weigh it on the balance Estimate volume of fish gelatin by eye Dissolve sucrose in water then add to fish gelatin afterwar

WHOLEMOUNT IMMUNO Fix tadpoles overnight @ 4 ° C in 4% Paraformaldehyde in 0.1M P.B. (aliquots in freezer) Rinse in 0.1M Phosphate Buffer Dissect out brains Pre-incubate in blocking solution in cold room on shaker or at room temperature for ~ 30 min Drain and replace with 1 ° antibody solution Incubate for 24hours in cold room on shaker Wash 3 x 30 min with blocking solution Wash overnight in blocking solution (4 th wash…) Drain and add 2 ° antibody solution Incubate overnight in cold room on shaker Rinse 3 x 30 min in phosphate buffer wash overnight in phosphate buffer (4 th wash…) Place brains on slide (Opening of 4 th ventricle indicates dorsal side) Mount in DAKO cytomation mounting medium using insect pins as spacers Seal slides with nail polish – we didn’t quite do this! Recipes: Blocking Solution 0.3 % triton-x-100 5 % Normal Goat Serum (or goat seru

  4% Paraformaldehyde in 0.1M Phosphate Buffer. (Use special glassware for fix.)  To make 500mL: 1) Heat milli-Q water (200mL) to about 80 C using water bath.  In hood: 2) Place hot water from step 1 on a stir plate. 3) Add 20 g paraformaldehyde crystals while stirring. 4) Add drops (about 20) of 1.0N NaOH slowly until paraformaldehyde is well dissolved. 5)   Add 250mL 0.2M sodium phosphate buffer      (210mL dibasic and 40mL monobasic). 6)   Transfer to graduated cylinder and add milli-Q water to get a final volume of 500mL. 7)   Test pH of a small aliquot using pH test paper.   Adjust to approx pH 7.4. 8)   Filter and store in a fixative fridge.

Prepare the following solutions: (To determine the amount needed for your experiment, please note that one tube of Solution 1 will yield a total of 100mL of working solution, at a final concentration of 0.5ug/mL.) Solution 1*: 5 μL DAPI (10 mg/ml solution) 995 μL PB * Solution is viable for one month if stored at 4° in the dark Solution 2 (working solution): 10 μL of Solution 1 990 μL PB Treatment: →Add Solution 2 (working solution) to your tissue/cells and incubate at room temperature for 1- 5 minutes. →Rinse 2X with PB. →Mount coverslip with Shur/Mount, Prolong or comparable antifade medium. →Seal coverslips (if necessary) with Valap or nail polish.** **Try to avoid nail polish if are visualizing GFP, as the additives in nail polish tend to quench the signal.

1. Incubate 2 x 20 min. in 0.1M PB at R.T. 2. Incubate 4 x 10 min in 0.1M PB + 0.3% triton at R.T. 3. Incubate 30 min in 0.1M PB + 0.3% triton +5% NGS at RT 4. Incubate overnight with primary antibody _______________ Diluted _____________ in 0.1M PB + 0.03% triton at 40C. 5. Rinse 4 x 10 min in 0.1M PB + 0.3% triton at RT 6. Incubate slides 1 hr in secondary antibody _______________ Diluted _____________ in 0.1M PB + 0.03% triton at R.T. 7. Rinse 3 x. 10 min in 0.1M PB at R.T. 8. 3 drops mounting medium + coverslip. Store at –200C

Internal Saline Weight per 10 ml (mg) Mw Final Conc. (mM) Cs Meth Sul 182.40 228.0 90 MgCl 2 10.17 203.3 5 TEA-Cl 33.14 165.7 20 EGTA 38.04 380.4 10 HEPES 47.66 238.3 20 ATP 11.00 551.1 2 GTP 1.57           523.2   0.3 pH= 7.2   (7.15-7.25)                   (use CsOH) Osmolarity 250                            (use H2O) start with 8ml then fill up to 10 ml Aliquot 500 m l/ tube, then store at –80C for up to 1 month. External Saline Weight per 1 L* (mg) Mw Final Conc. NaCl 6720.6 58.44 115 mM KCl 298.2 74.55 4 mM CaCl 2 -2H 2 O 441 147.00 3 mM MgCl 2 -6H 2 O 610 203.30 3 mM HEPES 1191.5