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How long can you effectively store a glycerol stock at -20 degrees Celsius?

How long can you effectively store a glycerol stock at -20 degrees Celsius?


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I know that glycerol stocks are typically kept in a -80 °C freezer, however there are some people who do not have access to such equipment. How long would you be able to keep a glycerol stock at around -20 °C (typical household freezer temperature). I would like to create a new stock before anything goes amiss.


While I believe that it is possible to keep a bacterial culture viable while frozen in glycerol stock at around -20 °C, as attested to by WYSIWYG, it is important to note that your "typical household freezer" may not be suitable for such a purpose.

This is because household freezers usually have an "auto-defrost" function. This makes it temporarily thaw out to remove the frost that condenses from atmospheric moisture. If storing sensitive materials such as bacterial/cell culture frozen stocks, this would lead to repeated freeze-thaw cycles which would damage the viability of the stocks.

For that reason, almost all laboratory freezers do not have that function, and instead need to be manually defrosted routinely.


How to Store Your Concentrated Proteins

Like graduate students, proteins are sensitive to rough handling. This is particularly true when they (the proteins, not the students!) are being concentrated, purified, and stored. We’ve covered the many options out there for concentrating your proteins, along with how to handle protein extracts to keep your proteins safe from degradation.

But proteins can degrade even after you’ve extracted and concentrated them! Storage itself can lead to protein breakdown, aggregation, and inactivation. And while there are many storage options, each has its own tradeoffs. Therefore, to help keep your proteins safe during storage, we’ll give you an overview of storage options along with six quick, handy tips!


Question on bacterial culture - (Sep/28/2006 )

A starter 5ml bacterial culture is inoculated for 24 hrs. If i want to stop here for a while before going for larger culture/miniprep, how long can i keep this 5ml culture in 4oC?

if the antibiotics have not been exhausted by the 24hr incubation, if your sterile technique is okay and there is no contamination, if the 5ml culture is properly covered, and if the 4 Celsius cool room/fridge is clean and not producing tons of spores, and nobody tips the culture on the floor.

The 5ml culture will probably last 1 month. I wouldn't keep it for anymore then 2. (Although I have labmate who do keep their culture for 6months)

If you want to miniprep it for plasmid, you can spin the cells down and freeze the pellet at -20 degrees. To keep the culture for subculturing, then I agree with perneseblue, make sure there is no chance of contamination.

if possible, you'd better dilute your starter culture 1/500 or 1/1000 into selective LB medium(according to your midi prep protocol), grow for 12-16hrs. and centrifuge to harvest bacterial cells at conditions stated in your midi kit protocol. after removing supernatant, you can freeze your abcterial cell pellet at -20C for later use. this is where i usually stop during midiprep

for storage, I would streak it onto a plate, grow it O/N, then parafilm it and put at 4C for up to a month

when you are inoculating with an overnight culture, you need a healthy, heavy growth. I do not think it's wise to leave it sitting at 4C for weeks and then try to re-use it. there is a reason we use overnight cultures to spike larger batches.


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Contents

Although achiral, glycerol is prochiral with respect to reactions of one of the two primary alcohols. Thus, in substituted derivatives, the stereospecific numbering labels the molecule with a "sn-" prefix before the stem name of the molecule. [8] [9] [10]

Glycerol is generally obtained from plant and animal sources where it occurs in triglycerides, esters of glycerol with long-chain carboxylic acids . The hydrolysis, saponification, or transesterification of these triglycerides produces glycerol as well as the fatty acid derivative:

Triglycerides can be saponified with sodium hydroxide to give glycerol and fatty sodium salt or soap.

Typical plant sources include soybeans or palm. Animal-derived tallow is another source. Approximately 950,000 tons per year are produced in the United States and Europe 350,000 tons of glycerol were produced per year in the United States alone from 2000 to 2004. [11] The EU directive 2003/30/EC set a requirement that 5.75% of petroleum fuels are to be replaced with biofuel sources across all member states by 2010. It was projected in 2006 that by the year 2020, production would be six times more than demand, creating an excess of glycerol. [7]

Glycerol from triglycerides is produced on a large scale, but the crude product is of variable quality, with a low selling price of as low as 2-5 U.S. cents per kilogram in 2011. [12] It can be purified, but the process is expensive. Some glycerol is burned for energy, but its heat value is low. [13]

Crude glycerol from the hydrolysis of triglycerides can be purified by treatment with activated carbon to remove organic impurities, alkali to remove unreacted glycerol esters, and ion exchange to remove salts. High purity glycerol (> 99.5%) is obtained by multi-step distillation a vacuum chamber is necessary due to its high boiling point (290 °C). [7]

Synthetic glycerol Edit

Although usually not cost-effective, glycerol can be produced by various routes from propene. The epichlorohydrin process is the most important: it involves the chlorination of propylene to give allyl chloride, which is oxidized with hypochlorite to dichlorohydrins, which reacts with a strong base to give epichlorohydrin. This epichlorohydrin is then hydrolyzed to give glycerol. Chlorine-free processes from propylene include the synthesis of glycerol from acrolein and propylene oxide. [7]

Because of the large-scale production of biodiesel from fats, where glycerol is a waste product, the market for glycerol is depressed. Thus, synthetic processes are not economical. Owing to oversupply, efforts are being made to convert glycerol to synthetic precursors, such as acrolein and epichlorohydrin. [14] (See the Chemical intermediate section of this article).

Food industry Edit

In food and beverages, glycerol serves as a humectant, solvent, and sweetener, and may help preserve foods. It is also used as filler in commercially prepared low-fat foods (e.g., cookies), and as a thickening agent in liqueurs. Glycerol and water are used to preserve certain types of plant leaves. [15] As a sugar substitute, it has approximately 27 kilocalories per teaspoon (sugar has 20) and is 60% as sweet as sucrose. It does not feed the bacteria that form a dental plaque and cause dental cavities. [ citation needed ] As a food additive, glycerol is labeled as E number E422. It is added to icing (frosting) to prevent it from setting too hard.

As used in foods, glycerol is categorized by the U.S. Academy of Nutrition and Dietetics as a carbohydrate. The U.S. Food and Drug Administration (FDA) carbohydrate designation includes all caloric macronutrients excluding protein and fat. Glycerol has a caloric density similar to table sugar, but a lower glycemic index and different metabolic pathway within the body, so some dietary advocates [ who? ] accept glycerol as a sweetener compatible with low-carbohydrate diets.

It is also recommended as an additive when using polyol sweeteners such as erythritol and xylitol which have a cooling effect, due to its heating effect in the mouth, if the cooling effect is not wanted. [16]

Medical, pharmaceutical and personal care applications Edit

Glycerin is mildly antimicrobial and antiviral and is an FDA approved treatment for wounds. The Red Cross reports that an 85% solution of glycerin shows bactericidal and antiviral effects, and wounds treated with glycerin show reduced inflammation after roughly 2 hours. Due to this it is used widely in wound care products, including glycerin based hydrogel sheets for burns and other wound care. It is approved for all types of wound care except third degree burns, and is used to package donor skin used in skin grafts. There is no topical treatment approved for third degree burns, and so this limitation is not exclusive to glycerin. [17]

Glycerol is used in medical, pharmaceutical and personal care preparations, often as a means of improving smoothness, providing lubrication, and as a humectant.

Ichthyosis and xerosis have been relieved by the topical use of glycerin. [18] [19] It is found in allergen immunotherapies, cough syrups, elixirs and expectorants, toothpaste, mouthwashes, skin care products, shaving cream, hair care products, soaps, and water-based personal lubricants. In solid dosage forms like tablets, glycerol is used as a tablet holding agent. For human consumption, glycerol is classified by the U.S. FDA among the sugar alcohols as a caloric macronutrient. Glycerol is also used in blood banking to preserve red blood cells prior to freezing.

Glycerol is a component of glycerin soap. Essential oils are added for fragrance. This kind of soap is used by people with sensitive, easily irritated skin because it prevents skin dryness with its moisturizing properties. It draws moisture up through skin layers and slows or prevents excessive drying and evaporation. [ citation needed ]

Taken rectally, glycerol functions as a laxative by irritating the anal mucosa and inducing a hyperosmotic effect, [20] expanding the colon by drawing water into it to induce peristalsis resulting in evacuation. [21] It may be administered undiluted either as a suppository or as a small-volume (2–10 ml) enema. Alternatively, it may be administered in a dilute solution, e.g., 5%, as a high volume enema. [22]

Taken orally (often mixed with fruit juice to reduce its sweet taste), glycerol can cause a rapid, temporary decrease in the internal pressure of the eye. This can be useful for the initial emergency treatment of severely elevated eye pressure. [23]

Glycerol has also been incorporated as a component of bio-ink formulations in the field of bioprinting. [24] The glycerol content acts to add viscosity to the bio-ink without adding large protein, carbohydrate, or glycoprotein molecules.

Botanical extracts Edit

When utilized in "tincture" method extractions, specifically as a 10% solution, glycerol prevents tannins from precipitating in ethanol extracts of plants (tinctures). It is also used as an "alcohol-free" alternative to ethanol as a solvent in preparing herbal extractions. It is less extractive when utilized in a standard tincture methodology. Alcohol-based tinctures can also have the alcohol removed and replaced with glycerol for its preserving properties. Such products are not "alcohol-free" in a scientific or FDA regulatory sense, as glycerol contains three hydroxyl groups. Fluid extract manufacturers often extract herbs in hot water before adding glycerol to make glycerites. [25] [26]

When used as a primary "true" alcohol-free botanical extraction solvent in non-tincture based methodologies, glycerol has been shown to possess a high degree of extractive versatility for botanicals including removal of numerous constituents and complex compounds, with an extractive power that can rival that of alcohol and water–alcohol solutions. [27] That glycerol possesses such high extractive power assumes it is utilized with dynamic (i.e. critical) methodologies as opposed to standard passive "tincturing" methodologies that are better suited to alcohol. Glycerol possesses the intrinsic property of not denaturing or rendering a botanical's constituents inert like alcohols (i.e. ethyl (grain) alcohol, methyl (wood) alcohol, etc.) do. Glycerol is a stable preserving agent for botanical extracts that, when utilized in proper concentrations in an extraction solvent base, does not allow inverting or mitigates [Redox|reduction-oxidation]] of a finished extract's constituents, even over several years. [ citation needed ] Both glycerol and ethanol are viable preserving agents. Glycerol is bacteriostatic in its action, and ethanol is bactericidal in its action. [28] [29] [30]

Electronic cigarette liquid Edit

Glycerin, along with propylene glycol, is a common component of e-liquid, a solution used with electronic vaporizers (electronic cigarettes). This glycerol is heated with an atomizer (a heating coil often made of Kanthal wire), producing the aerosol that delivers nicotine to the user. [31]

Antifreeze Edit

Like ethylene glycol and propylene glycol, glycerol is a non-ionic kosmotrope that forms strong hydrogen bonds with water molecules, competing with water-water hydrogen bonds. This interaction disrupts the formation of ice. The minimum freezing point temperature is about −36 °F (−38 °C) corresponding to 70% glycerol in water.

Glycerol was historically used as an anti-freeze for automotive applications before being replaced by ethylene glycol, which has a lower freezing point. While the minimum freezing point of a glycerol-water mixture is higher than an ethylene glycol-water mixture, glycerol is not toxic and is being re-examined for use in automotive applications. [32] [33]

In the laboratory, glycerol is a common component of solvents for enzymatic reagents stored at temperatures below 0 °C due to the depression of the freezing temperature. It is also used as a cryoprotectant where the glycerol is dissolved in water to reduce damage by ice crystals to laboratory organisms that are stored in frozen solutions, such as fungi, bacteria, nematodes, and mammalian embryos.

Chemical intermediate Edit

Glycerol is used to produce nitroglycerin, which is an essential ingredient of various explosives such as dynamite, gelignite, and propellants like cordite. Reliance on soap-making to supply co-product glycerol made it difficult to increase production to meet wartime demand. Hence, synthetic glycerol processes were national defense priorities in the days leading up to World War II. Nitroglycerin, also known as glyceryl trinitrate (GTN) is commonly used to relieve angina pectoris, taken in the form of sub-lingual tablets, patches, or as an aerosol spray.

An oxidation of glycerol affords mesoxalic acid. [34] Dehydrating glycerol affords hydroxyacetone.

Vibration damping Edit

Glycerol is used as fill for pressure gauges to damp vibration. External vibrations, from compressors, engines, pumps, etc., produce harmonic vibrations within Bourdon gauges that can cause the needle to move excessively, giving inaccurate readings. The excessive swinging of the needle can also damage internal gears or other components, causing premature wear. Glycerol, when poured into a gauge to replace the air space, reduces the harmonic vibrations that are transmitted to the needle, increasing the lifetime and reliability of the gauge. [35]

Niche uses Edit

Film industry Edit

Glycerol is used by the film industry when filming scenes involving water to stop areas from drying out too quickly. [36]

Glycerine is used—combined with water (around in a 1:99 proportion)—to create a smooth smoky environment. The solution is vaporized and pushed into the room with a ventilator.

Ultrasonic couplant Edit

Glycerol can be sometimes used as replacement for water in ultrasonic testing, as it has favourably higher acoustic impedance (2.42MRayl vs 1.483MRayl for water) while being relatively safe, non-toxic, non-corrosive and relatively low cost. [37]

Internal combustion fuel Edit

Glycerol is also used to power diesel generators supplying electricity for the FIA Formula E series of electric race cars. [38]

Research on uses Edit

Research has been conducted to produce value-added products from glycerol obtained from biodiesel production. [39] Examples (aside from combustion of waste glycerol):

    gas production [40] is a potential fuel additive. [41]
  • Glycerol is one of the most used additive for starch thermoplastic. [42][43]
  • Conversion to propylene glycol[44]
  • Conversion to acrolein[45][46]
  • Conversion to ethanol[47]
  • Conversion to epichlorohydrin, [48] a raw material for epoxy resins

Glycerol is a precursor for synthesis of triacylglycerols and of phospholipids in the liver and adipose tissue. When the body uses stored fat as a source of energy, glycerol and fatty acids are released into the bloodstream.

Glycerol is mainly metabolized in the liver. Glycerol injections can be used as a simple test for liver damage, as its rate of absorption by the liver is considered an accurate measure of liver health. Glycerol metabolism is reduced in both cirrhosis and fatty liver disease. [49] [50]

Blood glycerol levels are highly elevated during diabetes, and is believed to be the cause of reduced fertility in patients who suffer from diabetes and metabolic syndrome. Blood glycerol levels in diabetic patients average three times higher than healthy controls. Direct glycerol treatment of testes has been found to cause significant long-term reduction in sperm count. Further testing on this subject was abandoned due to the unexpected results, as this was not the goal of the experiment. [51]

Circulating glycerol does not glycate proteins as do glucose or fructose, and does not lead to the formation of advanced glycation endproducts (AGEs). In some organisms, the glycerol component can enter the glycolysis pathway directly and, thus, provide energy for cellular metabolism (or, potentially, be converted to glucose through gluconeogenesis).

Before glycerol can enter the pathway of glycolysis or gluconeogenesis (depending on physiological conditions), it must be converted to their intermediate glyceraldehyde 3-phosphate in the following steps:


What is the shelf life of all three vaccines?

The Pfizer vaccine’s lifespan depends largely on the temperature at which it is stored. All Pfizer vaccines will be shipped in special thermal shippers, which will maintain a temperature of -70 degrees Celcius, and can be used as temporary storage units by refilling them with dry ice whenever necessary. The vaccine can be stored in these containers for up to 30 days.

Stored in the refrigerators commonly found in hospitals and pharmacies, the vaccine can last for five days, assuming temperatures remain between 2-8 degrees Celsius. This can give COVID vaccines stored in the temporary containers and then shifted to refrigerators a shelf life of 35 days. In mid-May 2021, the European Medicines Agency said Pfizer doses could be refrigerated for 30 days instead of the original five.

At “ultra-low temperatures,” doses of the Pfizer vaccine can last up to six months. Once thawed, however, vaccines cannot be refrozen and stored. To ensure that no vaccines are stored improperly and lose their viability, Pfizer intends to utilize “GPS-enabled thermal sensors,” which will track the temperature of each shipment and allow Pfizer to “proactively prevent unwanted deviations and act before they happen,” according to the Pfizer website.

In late February, the FDA said the Pfizer vaccine, which was approved for kids aged 12-15 in mid-May, could be stored at standard freezer temperatures for up to two weeks. A few months later, Pfizer CEO Pfizer CEO Albert Bourla said the company was working on a new version of the vaccine that could be stored for six months in normal refrigerator temperatures.

The Moderna vaccine similarly depends on freezing temperatures to maintain viability. The vaccine must be kept at the same temperature as the Pfizer vaccine: between 2-8 degrees Celsius (or 36-46 degrees Fahrenheit). It will remain stable at these temperatures for three months (originally, the company said it could only last 30 days at those temperatures). At room temperature, the vaccine will remain viable for up to 12 hours.

At freezing temperatures, the Moderna vaccine can be stored for up to six months.

If the vaccines are thawed and then can’t be used in their current location, though, states are trying to relocate those doses so they can be given to people before they expire. Either that or people who wouldn’t be a priority patient have scored the vaccine for being in the right place at the right time.

During the Texas winter storms in mid-February, a power outage shut down a facility storing more than 8,000 Moderna vaccine doses. Officials managed to get more than 5,000 people vaccinated during the next 12 hours, and for the remainder of the doses, Moderna gave specific guidance so they could be re-refrigerated and kept safe.

Meanwhile, the Johnson & Johnson vaccine, which was approved for the U.S. in February and which requires only a single dose, doesn’t require ultra-freezing temperatures to store. According to Johnson & Johnson, the vaccine “is compatible with standard vaccine storage and distribution channels with ease of delivery to remote areas. The vaccine is estimated to remain stable for two years at -4°F (-20°C), and a maximum of three months at routine refrigeration at temperatures of 36-46°F (2 to 8°C).

But if vaccines are thawed and then expire before they find somebody’s arm—and that was a big worry in Ohio in June when 200,000 Johnson & Johnson doses were set to expire—here’s what happens to the unused doses.

By mid-June 2021, with a number of states worried that their Johnson & Johnson doses would expire before they could be used, Dr. Anthony Fauci said the FDA was trying to determine if the expiration dates could be adjusted or extended.


How Will These Storage Demands Be Met?

Keating anticipates that these requirements will significantly complicate the distribution of BNT162b2. In order to ensure the efficacy of the vaccine, according to Keating, people will need to be vaccinated at “centralized locations with access to minus 80 degrees Celsius freezers” or dry ice containers.

But this equipment is high maintenance in and of itself. Dry ice containers need “to be replenished regularly and dry ice supply may prove to be difficult to maintain,” Keating says.

Pfizer has preempted criticism of BNT162b2’s design by developing and manufacturing storage units specifically tailored to the vaccine. Roughly the size of a suitcase, these units can carry at least 975 doses and are packed with enough dry ice “to recharge it one more time,” Jessica Atwell, PhD, assistant scientist in the division of global disease epidemiology and control in the department of international health at the Johns Hopkins Bloomberg School of Public Health, tells Verywell.

However, it won’t be feasible to ship them worldwide.

“Doing that in high-income countries like the U.S. is one thing," Atwell says. "Trying to do that in low- and middle-income countries around the world where even a normal 2 to 8 degrees C, refrigerator-like temperature, can be really difficult in many parts of the world. So it's definitely an implementation challenge.”

Perhaps the biggest barrier to the widespread distribution of a vaccine that needs to be kept as cold as BNT162b2: There’s no precedent for it. “We don’t currently use any [vaccines] that require minus 70-degree storage,” Atwell says.


3. Protein-protein and protein-DNA interactions

An important task in deciphering protein function is the identification of other entities with which it interacts. Although C. elegans has not been exploited as an organism for biochemical analysis it is clearly amenable to such studies. Below are protocols describing immunoprecipitation (IP) and chromatin immunoprecipitation (ChIP) that should serve as general guidelines for in vivo interaction studies in C. elegans . These interactions can often be confirmed by standard in vitro techniques such as two-hybrid, GST pull-down studies, and electrophoresis mobility shift assays (EMSA).

Protocol 16: Immunoprecipitation from embryo lysates (Ray Chan and Barbara Meyer)

Seeding asynchronous liquid worm culture

Float adults worms off 9-cm NGM plates with 5 ml of M9. Approximately 6𔃆 plates saturated with asynchronous population of worms, but not starved, will be needed to seed each liter of liquid culture.

Add 10󈝻 ml of saturated HB101 and monitor the food supply at least once per day. Spot 1 drop of culture onto a 5-cm NGM plate and allow the liquid to evaporate for about 1𔃀 minutes until the worms can crawl. If there is sufficient food, the worms should not scatter or forage. Alternatively, starved worms appear somewhat translucent.

Harvesting asynchronous culture and seeding synchronous cultures

Filter culture through a 35-μm miracloth (Calbiochem) to collect gravid hermaphrodites.

Wash with approximately 0.5 L of dH2O.

Treat each liter of culture with 100 ml of freshly made alkaline-bleach solution. Mix on a stir-plate for 5󈝶 minutes (for mutant worms, it may be advisable to bleach for less than 5 minutes). Stop the bleaching process when the adult worms start to break open.

Alkaline-bleach solution (100 mL):
75 mL H2O
20 mL commercial bleach
5 mL 10 N NaOH

Weigh a 250-mL conical centrifuge tube. Record weight __________ g.

Centrifuge the bleached worms at 1𔃀,000 rpm in a tabletop centrifuge. Stop the centrifuge as soon as the speed reaches 2,000 rpm. (It takes several minutes for the rotor to come to a complete stop).

Resuspend the worm in M9 and centrifuge as in step 5. Repeat this wash step once more for a total of two M9 washes.

Weigh the centrifuge tube with the washed embryos.

Combined weight of centrifuge tube and embryos __________ g.

Weight of the embryos __________ g.

Seed 0.5 to 2 grams of embryos per 1-L completed S-basal medium. Generally use 0.5𔂿 g for N2 and 1𔃀 g for mutant worms. Allow the embryos to hatch overnight without food and feed the synchronized L1 larvae the next day.

Wash unused embryos in homogenization buffer, resuspend in 1 mL of homogenization buffer per gram of embryo and store in − 80°C freezer.

All steps are performed at 4°C.

Thaw 10󈝻 mL of embryos frozen in homogenization buffer.

Place the tube on ice and sonicate it with ten 30-second bursts at 15% power on a Heat System XL2020 sonicator with a standard tapered microtip (cat.#419, 3 mm diameter). Wait 1 minute in between bursts for cooling.

Pellet debris by centrifugation at 6,500 rpm (5,000 × g) in a SS-34 rotor for 20 minutes. Collect the supernatant in a clean 50-mL tube.

Sonicate as described in step 2 to shear the DNA. Pellet debris by centrifugation at 14,500 rpm (25,000 × g) in a SS-34 rotor for 20 minutes. Collect and quick-freeze the supernatant in 0.5𔂿 mL aliquots.

All steps performed at 4°C.

Thaw embryo lysates on ice. Incubate lysates with Protein A Sepharaose beads (100 μL per mL lysates Amersham) or IgGsorb (100 μL per mL lysates The Enzyme Center) for 10󈞊 minutes. Spin in a microcentrifuge for 2 min. at 500 × g to pellet the Sepharose beads or IgGsorb. Transfer the supernatant to a new microfuge tube and spin in a microcentrifuge at top speed for 10 min. Use the supernatant for IP.

Incubate approximately 5 μg of affinity purified antibodies with 3 mg total protein of embryo lysates for 2 hrs. Bring the final volume up to 1.0 to 1.4 mL using ChIP buffer with 140 mM KCl.

Pellet non-specific precipitates by spinning in a microcentrifuge at top speed (approximately 16,000 × g ) for 10 min.

Transfer the supernatant to a new microfuge tube with 25 μL of Protein A Sepharose and place on a rocker/nutator for 30 min.

Pellet the antibody-antigen complexes captured on the Protein A Sepharose beads by spinning in a microcentrifuge for 2 min. at 500 × g . Remove the supernatant.

Wash the beads by adding 1 mL of ChIP buffer and spinning in a microcentrifuge for 2 min. at 500 × g . Remove the wash buffer. Repeat this step for a total of four washes.

Elute by boiling the beads with 1 x SDS sample buffer and loading directly onto an SDS-PAGE gel. Alternatively, incubate the beads with 200 μL of 0.1 M glycine (pH 3.0) at room temp. Pellet the beads as described above, remove and save the supernatant. Precipitate the eluate with trichloroacetic acids.

For buffer recipes see the Chromatin Immunoprecipitation protocol below.

Protocol 17: Chromatin Immunoprecipitation From Embryo Lysates (Ray Chan and Barbara Meyer)

Harvest embryos by bleaching gravid hermaphrodites. [A typical yield for N2 is 2𔃃 grams from 25 to 30 9-cm NGM plates].

Wash the embryos extensively with 1x M9 to remove the alkaline bleach solution.

Prepare 100 mL of formaldehyde solution [1 x M9 solution with 2% (v/v) formaldehyde]

Wash the embryos once in the formaldehyde solution. Aspirate away the wash solution. Add fresh formaldehyde solution to 50 mL and gently shake (using a nutator) at room temp for 30 minutes.

Wash the cross-linked embryos once with 50 mL of 0.1 M Tris-HCl (pH 7.5), followed by two 50-mL washes of 1x M9.

Wash the embryos once with homogenization buffer. Add 1 mL of homogenization buffer per gram embryo (based on the starting amount), quick-freeze and store at − 80°C.

Thaw 10󈝻 mL of cross-linked embryos and add fresh protease inhibitors.

Place the tube on ice and sonicate it with ten 30-second bursts at 15% power on a Heat System XL2020 sonicator with a standard tapered microtip (cat.#419, 3 mm diameter). Wait 1 minute in between bursts for cooling.

Pellet debris by centrifugation at 6,500 rpm (5,000 × g) in a SS-34 rotor for 20 minutes. Collect the supernatant in a clean 50-mL tube.

Sonicate as described in step 2 to shear the DNA. Pellet debris by centrifugation at 14,500 rpm (25,000 × g) in a SS-34 rotor for 20 minutes. Collect and quick-freeze the supernatant in 0.5𔂿 mL aliquots.

Thaw embryo lysates on ice. Incubate lysates with Protein A Sepharaose beads (100 μL per mL lysates Amersham) or IgGsorb (100 μL per mL lysates The Enzyme Center) for 10󈞊 minutes. Spin in a microcentrifuge for 2 min. at 2,000 × g to pellet the Sepharose beads or IgGsorb. Transfer the supernatant to a new microfuge tube and spin in a microcentrifuge at top speed for 10 min. Use the supernatant for IP.

Incubate approximately 5 μg of affinity purified antibodies with 3 mg total protein of embryo lysates for 2 hrs.

Pellet non-specific precipitates by spinning in a microcentrifuge at top speed (approximately 16,000 × g ) for 10 min.

Transfer the supernatant to a new microfuge tube with 25 μL of Protein A Sepharose and place on a rocker/nutator for 30 min.

Pellet the antibody-antigen complexes captured on the Protein A Sepharose beads by spinning in a microcentrifuge for 2 min. at 500 × g . Remove the supernatant.

Wash the beads as follows:

2x 1-mL of ChIP buffer with 100 mM KCl

2x 1-mL of ChIP buffer with 1 M KCl

Add 200 μL elution buffer [10 mM Tris-HCl (pH8), 1% (w/v) SDS]. Spin to pellet the beads. Transfer eluate to a clean microfuge tube. Repeat elution step once more. Combine the eluates (400 μL total vol).

Add 16 μL of 5 M NaCl and heat overnight at 65°C to reverse the formaldehyde cross-links. [Use a PCR machine with a heated top or a Hybaid oven to avoid condensation at top of the tube].

Adding 8 μL of 0.5 M EDTA and 16 μL of 1 M Tris-HCl (pH 6.8). Mix. Digest proteins with 20 μg of proteinase K (Boehringer Mannheim) for 1 hour at 45°C.

Phenol-chloroform extractions. Add 10󈞀 μg of glycogen. Mix. EtOH precipitate. Resuspend in 100 μL TE.

Buffer Recipes

M9 buffer: 6 g Na2HPO4, 3 g KH2PO4, 5 g NaCl, 0.25 g of MgSO47H2O per liter. Autoclave.

1 M potassium citrate, pH 6.0: Per liter solution, add 268.8 g tripotassium citrate, 26.3 g citric acid monohydrate and adjust pH with KOH. Autoclave to sterilize.

Trace metals solution: Per liter solution, add 1.86 g Na2EDTA, 0.69 g FeSO4·7H2O, 0.2 g MnCl2·4H2O, 0.29 g ZnSO4·7H2O, 0.016g CuSO4. Autoclave to sterilize and store in the dark.

1 M potassium phosphate, pH 6.0: Per liter solution, dissolve 136 g KH2PO4 in 900 mL dH2O and adjust pH with KOH. Autoclave to sterilize.

10 x S basal medium: Per liter solution, add 59 g of NaCl, 500 mL of 1 M potassium phosphate (pH 6), 10 mL of cholesterol (5 mg/mL in EtOH). Autoclave to sterilize. [Note: the cholesterol will not go into solution].

Complete S medium: Per liter, add 100 mL of 10 x S basal medium, 10 mL 1 M potassium citrate (pH 6), 10 mL trace metals solution, 3 mL 1 M MgSO4, 3 mL 1 M CaCl2. Autoclave to sterilize.

Homogenization buffer: 50 mM HEPES-KOH, pH 7.6 1 mM EDTA 140 mM KCl 0.5% NP-40 10% glycerol. Add fresh protease inhibitors (aprotinin, pepstatin A, leupeptin, PMSF) and 5 mM DTT before use.

ChIP buffer: 50 mM HEPES-KOH, pH 7.6 1 mM EDTA 0.05% NP-40. Add KCl to the desired concentration. Add fresh protease inhibitors (aprotinin, pepstatin A, leupeptin, PMSF) and 1 mM DTT before use. [This buffer contains less NP-40 and no glycerol compared to the homogenization buffer].

Protocol 18: Chromatin immunoprecipitation (Johnathan Whetstine and Yang Shi)

This protocol is a modified version of a protocol from Upstate Biotechnologies (www.upstate.com).

Chromatin is isolated from 3×10 5 embryos/ChIP reaction, which equates to no less than 400 μg chromatin per immunoprecipitation (IP). There is no harm in scaling up for cleaner results, especially with poor antibodies.

Adult N2 worms grown on either HB101 or RNAi expressing bacteria are bleached and washed before being immersed and rotated in 1.5% Formaldehyde/M9 buffer for 30 minutes at 16 °C.

Embryos are washed extensively with M9 (at least 3 times) and gently centrifuged. Be careful not to rupture embryos.

Add warm chromatin SDS lysis solution (30 °C Upstate biotechnologies cat. # 20-163) to the embryos (minimum of 3×10 5 embryos per 200 μl solution) and dounce homogonize at least 30 times.

Place the slurry on ice for at least 20󈞊 minutes.

Combine the total amount of embryos or adults and sonicate 25 times per 2ml of extract used for an approximately 500bp smear (15 sec constant, 20% output on some sonicators). Do not over heat or allow to foam excessively. Keep on ice.

Centrifuge the samples, and keep the supernatant for DNA quantification before processing.

Aliquot the supernatant (200 μl) and dilute 10-fold into Dilution buffer (Upstate Biotechnologies cat. # 20-153).

Pre-clear each sample twice with 80 μl of ssDNA/protein A agarose beads from Upstate Biotechnologies (cat. # 16-157).

Incubate samples with the indicated antibody overnight at 4 °C with constant rotation.

The next morning add 60 μl of beads and incubate for 1 hour. Centrifuge and wash the beads once at room temperature with constant rotation with each of the following buffers (Note: You can make these buffers, see Upstate recipe, but I find that the beads are cleaner when these products are used): 1.0 ml High Salt Solution (Upstate Biotechnologies cat. # 20-155), 1.0 ml Low Salt buffer Solution (Upstate Biotechnologies cat. # 20-154), 1.0 ml LiCl Solution (Upstate Biotechnologies cat. # 20-156). After the last wash, the beads are washed three times with 1X TE, pH 8.0, which makes the beads slightly translucent.

Prepare and add fresh elution buffer (1%SDS and 0.1M NaHCO3 250 μl) to the beads at room temperature with constant agitation. Keep the supernatant and repeat this step once more.

Reverse the elution with 0.2 M NaCl for 4 hours at 65 °C.

Treat the samples with proteinase K (10 μl 0.5M EDTA, 20 μl 1M Tris-HCl, pH 6.5, and 2 μl 10mg/ml proteinase K) for 1 hour at 45 °C and then extract with phenol:chloroform:isoamyl alcohol.

Precipitate the extracted solution with 20 μg of yeast tRNA, and resuspend each sample in 50 μl warm 10 mM Tris-HCl, pH 8.0. The samples are now ready for PCR reactions.


Algal spent biomass—A pool of applications

A. Catarina Guedes , . F. Xavier Malcata , in Biofuels from Algae (Second Edition) , 2019

2.3.1 Phospholipids

PLs consist of fatty acids, and a phosphate-containing moiety attached to either glycerol or (the amino alcohol) sphingosine—thus resulting in compounds with fat-soluble and water-soluble regions that are ubiquitous in cell membranes. Glycerol-containing PLs include phosphatidic acid, phosphatidylcholine (PC), phophatidylethanolamine, phosphatidylinositol, and phosphatidylserine. Sphingomyelin (SPH)—a major PL, consisting of sphingosine and PC. PLs and choline have several benefits for human health, as depicted in Table 4 . The level of PLs in various red macroalgae varies from 10% to 21% of total lipids these are largely PC (62%–78%) and PG (10%–23%) [81] .

Dietary PLs act as natural emulsifiers, and as such they facilitate digestion and absorption of fatty acids, cholesterol and other lipophilic nutrients. Algal phopholipids appear to bear a number of advantages relative to fish oils, because they are more resistant to oxidation (rancidity), have higher contents of EPA and DHA, with superior bioavailability, and provide a wider spectrum of health benefits for humans and animals [16] .


ARTIFICIAL INSEMINATION TECHNIQUES

The technique of inseminating a cow is a skill requiring adequate knowledge, experience and patience. Improper AI techniques can negate all other efforts to obtain conception. Semen must be deposited within the tract of the cow at the best location and at the best time to obtain acceptable conception rates. Early methods of AI involved deposition of the semen in the vagina, as would occur in natural mating. Those methods are not satisfactory. Fertility is low and greater numbers of sperm are required. Another method which gained popularity was the "speculum" method. This method is easily learned, but proper cleaning and sterilizing of the equipment is necessary, making it more impractical to inseminate than with the rectovaginal technique which is the most widely used AI method today.

In the recto-vaginal technique a sterile, disposable catheter containing the thawed semen is inserted into the vagina and then guided into the cervix by means of a gloved hand in the rectum. The inseminating catheter is passed through the spiral folds of the cow's cervix into the uterus. Part of the semen is deposited just inside the uterus and the remainder in the cervix as the catheter is withdrawn. Expulsion of the semen should be accomplished slowly and deliberately to avoid excessive sperm losses in the catheter. The body of the uterus is short therefore, care should be taken not to penetrate too deeply which might cause physical injury. In animals previously inseminated, the catheter should not be forced through the cervix since pregnancy is a possibility. Since research data show little variation in conception rates when semen is placed in the cervix, uterine body or uterine horns, some people recommend incomplete penetration of the cervical canal and deposition of semen in the cervix.

The recto-vaginal technique is more difficult to learn and practice is essential for acceptable proficiency but the advantages make this method of insemination more desirable than other known methods. With practice, the skillful technician soon learns to thread the cervix over the catheter with ease. If disposable catheters are used and proper sanitation measures are followed, there is little chance of infection being carried from one cow to another.

Timing of Insemination for Maximum Conception

A frequent question concerning AI is: What time during estrus should cows be bred for greatest chance of conception? Since estrus may last from 10 to 25 hours there is considerable latitude in possible time of insemination. Much research work has been conducted on this subject.

Controlled investigations were conducted by Trim Berger and Davis at Nebraska in 1943. These and other studies show that conception rate is lower when cows are bred prior to mid estrus or later than 6 hours after cessation of estrus (standing heat in this case). Maximal conception is obtained when cows are inseminated between mid estrus and the end of standing estrus, with good results up to 6 hours after estrus.

Success in insemination timing is dependent upon a good heat detection program. In large herds, this means assigning individual responsibility for heat detection and a continued education program for labor. A successful heat detection program and subsequent proper timing of insemination will pay dividends in increasing reproductive efficiency.



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