Recipes for Media Reagents and Stock Solutions

Recipes For Media Reagents And Stock Solutions-Free PDF

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Silencing Genomes Recipes,1 M Tris pH 7 6,10 Ligation Buffer. 0 1 M Adenosine Triphosphate ATP,2 Ligation Buffer ATP. T tailing L4440 Vector,VII Bacterial Transformation. 1 M Calcium Chloride CaCl2,50 mM Calcium Chloride CaCl2. Competent Cell Preparation,VIII Plasmid Minipreparation.
0 5 M Ethylenediaminetetraacetic Acid EDTA,Tris EDTA TE Buffer. Glucose Tris EDTA GTE,5 M Potassium Acetate KOAc,Potassium Acetate Acetic Acid. 10 Sodium Dodecyl Sulfate SDS,1 SDS 0 2 N NaOH,Notes on Buffers. 1 Typically solid reagents are dissolved in a volume of de. ionized or distilled water equivalent to 70 80 of the finished. volume of buffer This leaves room for the addition of acid or. base to adjust the pH Then water is added to bring the. solution up to the final volume, 2 Buffers typically are used as 1 or 10 solutions Buffers are. diluted when mixed with other reagents to produce a working. concentration of 1, 3 The buffer supplied with the commercial enzymes used in.
these laboratories should be used unless noted otherwise. 4 Storage temperatures of 4 C and 20 C refer to normal. refrigerator and freezer non frost free temperatures. respectively, 5 The final concentration of each liquid reagent is given in the. right hand column of the reagent list, Dolan DNA Learning Center Cold Spring Harbor Laboratory 2. Silencing Genomes Recipes,I Bacterial Culture,4 N Sodium Hydroxide NaOH. Makes 100 mL,Store at room temperature indefinitely. 1 Slowly add 16 g of NaOH pellets m w 40 00 to 80 mL of. deionized or distilled water with stirring The solution will get. 2 When the NaOH pellets are completely dissolved add water to. a final volume of 100 mL,10 mg mL Ampicillin,Makes 100 mL.
Store at 20 C 1 year or 4 C 3 months, 1 Add 1 g of ampicillin sodium salt m w 371 40 to 100 mL of. deionized or distilled water in a clean 250 mL flask The. sodium salt dissolves readily however the free acid form is. difficult to dissolve,2 Stir to dissolve, 3 Pre wash a 0 45 or 0 22 m sterile filter Nalgene or Corning. by drawing through 50 100 mL of deionized or distilled water. Pass the ampicillin solution through the washed filter. 4 Dispense 10 mL aliquots in sterile 15 mL tubes Falcon 2059. or equivalent and freeze at 20 C,1 1 vol vol Distilled or Deionized Water Ethanol. Makes 100 mL,Store in a sealed container, 1 Add 50 mL of distilled or deionized water to 50 mL of 95 100. ethanol in a clean 200 mL bottle,12 5 mg mL Tetracycline.
Makes 80 mL,Store at 20 C 1 year or 4 C 3 months, 1 Add 1 0 g of tetracycline m w 444 44 to 75 mL of 1 1. vol vol distilled or deionized water ethanol in a clean 250 mL. 2 Stir to dissolve, 3 Bring up the volume to 80 mL with 1 1 vol vol distilled or. deionized water ethanol, 4 Pre wash a 0 45 or 0 22 m sterile filter Nalgene or Corning. by drawing through 50 100 mL of deionized or distilled water. Pass the tetracycline solution through the washed filter. Dolan DNA Learning Center Cold Spring Harbor Laboratory 3. Silencing Genomes Recipes, 5 Dispense 10 mL aliquots in sterile 15 mL tubes Falcon 2059. or equivalent wrap in aluminium foil tetracycline is light. sensitive and freeze at 20 C,Luria Bertani LB Broth.
Makes 1 liter,Store at room temperature indefinitely. 1 Weigh out,10 g of tryptone,5 g of yeast extract,10 g of NaCl m w 58 44. Alternatively use 25 g of premix containing all of these. ingredients, 2 Add all ingredients to a clean 2 liter flask that has been rinsed. with deionized or distilled water, 3 Add 1 liter of deionized or distilled water to flask. 4 Add 0 5 mL of 4 N NaOH, 5 Stir to dissolve the dry ingredients preferably using a.
magnetic stir bar, 6 If preparing for mid log cultures Split LB broth into two 500 mL. aliquots in 2 liter flasks Plug top with cotton or foam and. cover with aluminum foil Alternatively cover with aluminum. foil only Autoclave for 15 20 minutes at 121 C, If preparing for general use in transformations Dispense 100. mL aliquots into sterile 150 250 mL bottles using one of the. following methods, a Loosely put on the caps Autoclave for 15 20 minutes at. 121 C To help guard against breakage autoclave the. bottles in a shallow pan with a small amount of water. b Prewash a 0 45 or 0 22 m sterile filter Nalgene or. Corning by drawing through 50 100 mL of deionized or. distilled water Pass LB broth through the filter and. dispense aliquots into sterile bottles, NOTE LB broth can be considered sterile as long as the solution. remains clear Cloudiness is a sign of contamination by microbes. Always swirl the solution to check for bacterial or fungal cells that. may have settled at the bottom of the flask or bottle. Dolan DNA Learning Center Cold Spring Harbor Laboratory 4. Silencing Genomes Recipes,LB Broth Antibiotic,Makes 100 mL.
Store at 4 C 3 months, 1 Sterilely add 1 mL of stock antibiotic to 100 mL of cool LB. 2 Swirl to mix,LB Agar Plates,Makes 35 40 plates, Store at 4 C 3 months or room temperature 3 months. 1 Weigh out,10 g of tryptone,5 g of yeast extract,10 g of NaCl m w 58 44. 15 g of agar, Alternatively use 40 g of premix containing all of these. ingredients, 2 Add all ingredients to a clean 2 liter flask that has been rinsed.
with deionized or distilled water,3 Add 1 liter of deionized or distilled water. 4 Add 0 5 mL of 4 N NaOH, 5 Stir to dissolve dry ingredients preferably using a magnetic stir. bar Any undissolved material will dissolve during autoclaving. 6 Cover flask mouth with aluminum foil and autoclave solution. for 15 minutes at 121 C, 7 Allow the solution to cool just until the flask can be held by. bare hands 55 60 C If the solution cools too long and the. agar begins to solidify remelt by briefly autoclaving for 5. minutes or less or heating it in a microwave oven for a few. 8 While the agar is cooling mark culture plate bottoms with the. date and description of the media e g LB If using, presterilized polystyrene plates carefully cut the end of the. plastic sleeves and save the sleeves for storing the poured. plates Spread the plates out on the lab bench, 9 When the agar flask is cool enough to hold lift the lid of a.
culture plate only enough to pour the solution Do not place the. lid on the lab bench Quickly pour in enough agar to just cover. the bottom of the plate 25 30 mL Tilt the plate to spread. the agar evenly and immediately replace the lid, 10 Continue pouring agar into plates Occasionally flame the. mouth of the flask to maintain sterility, Dolan DNA Learning Center Cold Spring Harbor Laboratory 5. Silencing Genomes Recipes, 11 To remove bubbles in the surface of the poured agar touch. the plate surface with the flame from a Bunsen burner while. the agar is still liquid,12 Allow agar to solidify undisturbed. 13 If possible incubate the plates lidside down for several hours. at 37 C overnight if convenient This dries the agar limiting. condensation when plates are stored under refrigeration It. also allows the ready detection of any contaminated plates. 14 Stack plates in their original sleeves for storage. LB Agar Antibiotic,Makes 30 45 plates,Store at 4 C 3 months.
1 Follow recipe above for LB agar plates through Step 8. 2 When the agar flask is cool enough to hold sterilely add 10. mL of stock antibiotic Ampicillin and tetracycline are destroyed. by heat therefore it is essential to cool the agar before adding. the antibiotic, 3 Swirl the flask to mix the antibiotic and agar solution. 4 Resume recipe above at Step 9, NOTE In a pinch antibiotic containing plates can be made. quickly by evenly spreading 200 L of 10 mg mL antibiotic on the. surface of an LB agar plate Allow the agar to absorb the antibiotic. for 10 20 minutes before use Outdated antibiotic plates can be. refurbished in this manner, Dolan DNA Learning Center Cold Spring Harbor Laboratory 6. Silencing Genomes Recipes,II C elegans Culture,5 mg mL Cholesterol dissolved in 100 ethanol. Makes 10 mL,Store at 20 C 1 year or 4 C 3 months, 1 Add 50 mg of cholesterol to 10 mL of 100 ethanol in a sterile.
15 mL tube Falcon 2059 or equivalent,2 Stir to dissolve. 3 Freeze at 20 C,NGM lite Plates, Eric Lambie originally described NGM lite plates in Worm. Breeder s Gazette 13 5 11 February 1 1995 These plates. contain a simple complete medium for culturing C elegans. 1 Weigh out,2 g of NaCl,4 g of Bactotryptone,3 g of KH2PO4. 0 5 g of K2HPO4,20 g of agar, 1 mL of 5 mg mL cholesterol dissolved in 100 ethanol. 2 Add all ingredients to a clean 2 liter flask that has been rinsed. with deionized or distilled water,3 Add 1 liter of deionized or distilled water.
4 Stir to dissolve dry ingredients preferably using a magnetic stir. bar Any undissolved material will dissolve during autoclaving. 5 Cover the flask mouth with aluminum foil and autoclave the. solution for 15 minutes at 121 C, 6 After autoclaving cool this solution to 55 C in a water bath If. the solution cools too long and the agar begins to solidify melt. by briefly autoclaving for 5 minutes or less or heating it in a. microwave oven for a few minutes, 7 Pour the solution into 6 cm plastic petri dishes Add enough to. fill the plates about half full If the plate has large bubbles that. are large enough for worms to crawl into pop the bubbles by. briefly flaming the surface with a Bunsen burner Occasionally. flame the mouth of the flask to maintain sterility. 8 Allow the plates to cool for at least 1 day before seeding them. Dolan DNA Learning Center Cold Spring Harbor Laboratory 7. Silencing Genomes Recipes,1M Isopropyl D thiogalactoside IPTG. Makes 5 mL,Store at 20 C indefinitely,1 Add 1 19 g of Isopropyl D thiogalactoside IPTG. m w 238 3 to 5 mL of deionized or distilled water,in a clean 15 mL tube.
2 Mix or rock gently to dissolve,3 Pass the IPTG solution through a 0 45 or 0 22 m. sterile filter Nalgene or Corning,4 Dispense the solution into a sterile 15 mL tube. Falcon 2059 or equivalent and freeze at 20 C,NGM lite Ampicillin IPTG plates. 1 Follow recipe above for NGM lite plates through, 2 When the agar flask is cool enough to hold sterilely. add 10 mL of 10 mg mL ampicillin and 0 4 mL of,1M IPTG Ampicillin and IPTG are destroyed by.
heat therefore it is essential to cool the agar,before adding them. 3 Swirl the flask to mix the ampicillin and IPTG with. the agar solution,4 Resume NGM lite Plates recipe above at Step 8. Makes 1 liter,Store at room temperature indefinitely. 1 Add the following ingredients to 500 mL of deionized or distilled. water in a 1 liter bottle,3 g of KH2PO4,6 g of Na2HPO4. 5 g of NaCl,1 mL of 1M MgSO4, 2 Stir to dissolve preferably using a magnetic stir bar.
3 Add deionized or distilled water to bring total solution to 1 liter. 4 Ensure the bottle cap is loose and autoclave solution for 15. minutes at 121 C, 5 After autoclaving cool the solution to room temperature and. tighten the cap for storage, Dolan DNA Learning Center Cold Spring Harbor Laboratory 8. Silencing Genomes Recipes,Hypochlorite Solution,Makes 300 L. Make fresh daily,Mix in a 1 5 mL tube,200 L of 4 M NaOH. 300 L of bleach with 10 20 hypochlorite NaOCl,Freezing solution.
Makes 1 liter,Store at room temperature indefinitely. 1 Add the following ingredients to 500 mL of deionized or. distilled water in a 2 liter flask,5 85 g of NaCl,6 8 g of KH2PO4. 300 g of glycerol,5 6 mL of 1N NaOH, 2 Stir to dissolve preferably using a magnetic stir bar. 3 Add deionized or distilled water to bring total solution to 1 liter. 4 Ensure the bottle cap is loose and autoclave solution for 15. minutes at 121 C, 5 After autoclaving cool the solution to room temperature and. tighten the lid for storage, Dolan DNA Learning Center Cold Spring Harbor Laboratory 9.
Silencing Genomes Recipes,III Worm DNA Isolation,1 M Tris pH 8 3. Makes 100 mL,Store at room temperature indefinitely. 1 Dissolve 12 1 g of Tris Base m w 121 10 in 70,mL of deionized or distilled water. 2 Adjust the pH by slowly adding concentrated,hydrochloric acid HCl monitor with a pH meter. 5 M Potassium Chloride KCl,Makes 100 mL,Store at room temperature indefinitely.
1 Dissolve 37 275 g of KCl m w 74 55 in 70 mL of,deionized or distilled water. 2 Add deionized or distilled water to make a total. volume of 100 mL of solution,1 M Magnesium Chloride MgCl2. Makes 100 mL,Store at room temperature indefinitely. 1 Dissolve 20 3 g of MgCl2 6 hydrate m w,154 25 in 80 mL of deionized or distilled water. 2 Add deionized or distilled water to make a total. volume of 100 mL of solution,10 PCR Buffer,Makes 10 mL.
Store at 20 C indefinitely,Mix in a 15 mL tube,1 mL of 1 M Tris pH 8 3. 1 mL of 5 M KCl,0 15 mL of 1 M MgCl2,7 85 mL of deionized water. Dolan DNA Learning Center Cold Spring Harbor Laboratory 10. Silencing Genomes Recipes,IV Polymerase Chain Reaction. 1 Cresol Red Dye,Makes 50 mL,Store at room temperature indefinitely. Mix in a 50 mL tube,500 mg of cresol red dye,50 mL of distilled water.
Cresol Red Loading Dye,Makes 50 mL,Store at 20 C indefinitely. 1 Dissolve 17 g of sucrose in 49 mL of distilled water in a 50 mL. NGM lite plates 10 mg mL Ampicillin 1M Isopropyl D thiogalactoside IPTG NGM lite Ampicillin IPTG plates M9 Buffer Freezing Solution III Worm DNA Isolation 1 M Tris pH 8 3 5 M KCL 1 M Magnesium Chloride MgCl 2 10 PCR Buffer IV Polymerase Chain Reaction 1 Cresol Red Dye Cresol Red Loading Dye Primer Loading Dye Mix Primer Sequences

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