Molecular Cloning Protocol 28 s by Hybridization:Small Numbers MATERIALS 合O Media vectors and Ba 125 h METHOD n e ctnroypa a6n nd store it at 4'C in an inverted postion untl the results of the hvtridizatio REFERENCES e11e
Chapter:1 Protocol:28 Screening Bacterial Colonies by Hybridization: Small Numbers CHAPTER 1 > PROTOCOL 28 printer friendly version Protocol 28 Screening Bacterial Colonies by Hybridization: Small Numbers This procedure, a variant of the Grunstein and Hogness (1979) method, is used to screen a small number of bacterial colonies (<200) that are dispersed over several agar plates and are to be screened by hybridization to the same radiolabeled probe. The colonies are gridded onto a master plate and onto a nitrocellulose or nylon filter laid on the surface of a second agar plate. After a period of growth, the colonies on the filter are lysed and processed for hybridization. The master plate is stored until the results of the screening procedure become available. MATERIALS CAUTION: Please click for information about appropriate handling of materials. RECIPE: Please click for components of stock solutions, buffers, and reagents. Media Rich broth agar plates containing the appropriate antibiotic Rich broth agar plates containing chloramphenicol Additional Reagents Step 8 of this protocol requires the reagents listed in Chapter 1, Protocol 31 and Chapter 1, Protocol 32 . Vectors and Bacterial Strains E. coli strain, transformed with recombinant plasmids Use bacteria transformed by one of the methods described in Chapter 1, Protocol 23 , Chapter 1, Protocol 24 , Chapter 1, Protocol 25 and Chapter 1, Protocol 26 . E. coli strain, transformed with nonrecombinant plasmid (e.g., pUC, used as a negative control) METHOD 1. Place a nitrocellulose or nylon filter on an agar plate (test plate) containing the selective antibiotic. 2. Draw a numbered grid on a piece of graph paper (1-cm-square grid). Number the base of each agar master plate and place the plate on the grid. Draw a mark on the side of the plate at the 6 o'clock position. 3. Use sterile toothpicks or inoculating loops to transfer bacterial colonies one by one onto the filter on the test plate and then onto the master agar plate that contains the selective antibiotic but no filter. Make small streaks 2-3 mm in length (or dots) arranged according to the grid pattern under the dish. Streak each colony in an identical position on both plates. 4. Finally, streak a colony containing a nonrecombinant plasmid (e.g., pUC) onto the filter and the master plate. 5. Invert the plates and incubate them at 37°C until the bacterial streaks have grown to a width of 0.5-1.0 mm (typically 6- 16 hours). 6. Mark the filter in three or more asymmetric locations by stabbing through it and into the agar of the test plate with an 18- gauge needle, attached to a syringe, dipped in waterproof black drawing ink (India Ink). Mark the master plate in approximately the same locations. 7. Seal the master plate with Parafilm and store it at 4°C in an inverted position until the results of the hybridization reaction are available. 8. Lyse the bacteria adhering to the filter and bind the liberated DNA to the nitrocellulose or nylon filter using the procedures described in Chapter 1, Protocol 31 . Proceed with hybridization as described in Chapter 1, Protocol 32 . REFERENCES 1. Grunstein M. and Hogness D.S. 1975. Colony hybridization: A method for the isolation of cloned DNAs that contain a specific gene. Proc. Natl. Acad. Sci. 72:3961-3965. printer friendly version Buy The Book | Our Vision | Take The Tour | Newsletter | Search CSHL Press Home | Contact | Members Home | CSHL Home Copyright © 2000. Cold Spring Harbor Laboratory Press. http://www.molecularcloning.com/members/protocol.jsp?pronumber=28&chpnumber=1 [2002-2-18 16:15:47]
@片Molecular Cloning L A B O R A T O RY CHAPTER1>PROTOCOL ●irv Protocol 29 idization to an Media METHOD Lyse the ba 3 p.1.131o the print e the filter colony side up on the surface of a fresh LB (cr SOB)agar plate containing the REFERENCES e2e
Chapter:1 Protocol:29 Screening Bacterial Colonies by Hybridization: Intermediate Numbers CHAPTER 1 > PROTOCOL 29 printer friendly version Protocol 29 Screening Bacterial Colonies by Hybridization: Intermediate Numbers Bacterial colonies growing on agar plates are transferred en masse to nitrocellulose filters. The spatial arrangement of colonies on the plates is preserved on the filters. After transfer, the filters are processed for hybridization to an appropriate radiolabeled probe while the original (master) plate is incubated for a few hours to allow the bacterial colonies to regrow in their original positions. This technique, a variant of the Grunstein and Hogness (1975) method, was developed at Cold Spring Harbor Laboratory in 1975. The procedure works best with 90-mm plates containing <2500 colonies. MATERIALS CAUTION: Please click for information about appropriate handling of materials. RECIPE: Please click for components of stock solutions, buffers, and reagents. Media Rich broth agar plates (90-mm) containing appropriate antibiotics Rich broth agar plates containing chloramphenicol Additional Reagents Step 6 of this protocol requires the reagents listed in Chapter 1, Protocol 31 and Chapter 1, Protocol 32 . Vectors and Bacterial Strains E. coli strain, transformed with recombinant plasmids, as culture Use bacteria transformed by one of the methods described in Chapter 1, Protocol 23 , Chapter 1, Protocol 24 , Chapter 1, Protocol 25 and Chapter 1, Protocol 26 . METHOD 1. Plate out the transformed E. coli culture onto 90-mm LB or SOB agar plates, at dilutions calculated to generate up to 2500 transformed colonies. When the colonies reach an average size of 1.5 mm, transfer the plates from the incubator to a cold room. 2. Number the dry filters with a soft-lead pencil or a ball-point pen, wet them with water, and interleave them between dry Whatman 3MM filters. Wrap the stack of filters loosely in aluminum foil, and sterilize them by autoclaving (15 psi [1.05 kg/cm2] for 10 minutes on liquid cycle). 3. Place a dry, sterile detergent-free nitrocellulose filter, numbered side down, on the surface of the LB (or SOB) agar medium, in contact with the bacterial colonies (plated in Step 1), until it is completely wet. 4. Once the filter is in place, key the filter to the underlying medium by stabbing in three or more asymmetric locations through the filter with a 23-gauge needle attached to a syringe, dipped in waterproof black drawing ink. 5. Grip the edge of the filter with blunt-ended forceps and, in a single smooth movement, peel the filter from the surface of the agar. 6. Proceed with one of the following options as appropriate: • Lyse the bacteria adhering to the filter and bind the liberated DNA to the nitrocellulose or nylon filter using the procedures described in Chapter 1, Protocol 31 . Proceed with hybridization as described in Chapter 1, Protocol 32 . • Lyse the bacteria and immobilize the DNA as described in the alternative protocol on p. 1.131 of the print version of the manual. • Place the filter, colony side up, on the surface of a fresh LB (or SOB) agar plate containing the appropriate antibiotic. After incubation for a few hours, when the colonies have grown to a size of 2-3 mm, remove the filter and proceed with lysis and hybridization as described in Chapter 1, Protocol 31 and Chapter 1, Protocol 32 . • Amplify the colonies on the filter by transferring the filter to an agar plate containing chloramphenicol (170-200 µg/ml) and incubating for 12 hours at 37°C. Proceed with lysis and hybridization ( Chapter 1, Protocol 31 and Chapter 1, Protocol 32 ). • Use the filter to prepare a second replica: a. Place the filter colony side up on the surface of a fresh LB (or SOB) agar plate containing the appropriate antibiotic. b. Lay a dry nitrocellulose filter carefully on top of the first and key to it as described in Step 4 above. c. Incubate the "filter sandwich" for several hours at 37°C. d. Proceed with lysis and hybridization ( Chapter 1, Protocol 31 and Chapter 1, Protocol 32 ), keeping the filters as a sandwich during the lysis and neutralization steps, but peeling them apart before the final wash. 7. Incubate the master plate for 5-7 hours at 37°C until the colonies have regrown. Seal the plate with Parafilm, and store it at 4°C in an inverted position. REFERENCES 1. Grunstein M. and Hogness D.S. 1975. Colony hybridization: A method for the isolation of cloned DNAs that contain a specific gene. Proc. Natl. Acad. Sci. 72:3961-3965. printer friendly version Buy The Book | Our Vision | Take The Tour | Newsletter | Search CSHL Press Home | Contact | Members Home | CSHL Home Copyright © 2000. Cold Spring Harbor Laboratory Press. http://www.molecularcloning.com/members/protocol.jsp?pronumber=29&chpnumber=1 [2002-2-18 16:15:54]
Molecular Cloning CHAPTER1>PROTOCOL3 printer friendly versio Protocol 30 ies by Hybridization:Large Numbers MATERIALS Media csand%() %28出品 ade up.on the sur spreader tod cbagt-20rc ming trap mhe two f e the Petrl dish d top 3MM paper.and orient the two by making a series of holes with an 18 gthepprop nd25% REFERENCES 1.Hanahan D an Meselson M.1980.Plasmid screening at high colory density.Gene 10:63-67 ●printor triond山y vorsion
Chapter:1 Protocol:30 Screening Bacterial Colonies by Hybridization: Large Numbers CHAPTER 1 > PROTOCOL 30 printer friendly version Protocol 30 Screening Bacterial Colonies by Hybridization: Large Numbers This procedure is used to plate, replicate, and subsequently screen large numbers of bacterial colonies (up to 2 x 104 colonies per 150-mm plate or 104 colonies per 90-mm plate). MATERIALS CAUTION: Please click for information about appropriate handling of materials. RECIPE: Please click for components of stock solutions, buffers, and reagents. Media Rich broth agar plates containing appropriate antibiotics Plates that are 2-3 days old give the best results in this protocol because they absorb fluid from the bacterial inoculum more readily. Rich broth agar plates containing appropriate antibiotics and 25% (v/v) glycerol Rich broth agar plates containing chloramphenicol Additional Reagents Step 14 of this protocol requires the reagents listed in Chapter 1, Protocol 31 and Chapter 1, Protocol 32 . Vectors and Bacterial Strains E. coli strain, transformed with recombinant plasmids, as culture Use bacteria transformed by one of the methods described in Chapter 1, Protocol 23 , Chapter 1, Protocol 24 , Chapter 1, Protocol 25 and Chapter 1, Protocol 26 or Amplified aliquot of cDNA library, grown as culture Please see Chapter 11, Protocol 1. METHOD 1. Number the dry filters with a soft-lead pencil or a ball-point pen, wet them with water, and interleave them between dry Whatman 3MM filters. Wrap the stack of filters loosely in aluminum foil, and sterilize them by autoclaving (15 psi [1.05 kg/cm2] for 10 minutes on liquid cycle). 2. Use sterile, blunt-ended forceps to lay a sterile filter, numbered side down, on a 2-3-day-old LB (or SOB) agar plate containing the appropriate antibiotic. When the filter is thoroughly wet, peel it from the plate and replace it, numbered side up, on the surface of the agar. 3. Apply the bacteria, in a small volume of liquid, to the center of the filter on the surface of the agar plate. Use a sterile glass spreader to disperse the fluid evenly, leaving a border 2-3 mm wide around the circumference of the filter free of bacteria. 4. Incubate the plate (noninverted) with the lid ajar for a few minutes in a laminar flow hood to allow the inoculum to evaporate. Then close the lid, invert the plate, and incubate at 37°C until small colonies (0.1-0.2-mm diameter) appear (approx. 8-10 hours). 5. If desired, replica filters may be prepared at this stage (proceed with Step 6). Otherwise, prepare the bacterial colonies for storage at -20°C: a. Transfer the filter colony side up to a labeled LB (or SOB) agar plate containing the appropriate antibiotic and 25% glycerol. b. Incubate the plate for 2 hours at 37°C. c. Seal the plate well with Parafilm, and store it in an inverted position in a sealed plastic bag at -20°C. 6. Lay the master nitrocellulose or nylon filter colony side up on a sterile Whatman 3MM paper. 7. Number a damp, sterile nitrocellulose or nylon filter, and lay it on the master filter. Take care to prevent air bubbles from becoming trapped between the two filters. 8. Cover the filter sandwich with a second 3MM circle and place the bottom of a Petri dish on top of the 3MM paper. Press down firmly on the Petri dish with the palm of the hand to facilitate transfer of bacteria from the master filter to the replica. 9. Dismantle the Petri dish bottom and top 3MM paper, and orient the two filters by making a series of holes with an 18- gauge needle attached to a syringe. 10. Peel the filters apart. Lay the replica on a fresh LB (or SOB) agar plate containing the appropriate antibiotic. 11. Place the second replica filter (if made) and the master filter on a fresh LB (or SOB) agar plate containing the appropriate antibiotic and incubate all plates at 37°C until colonies appear (4-6 hours). 12. At this stage, when the bacteria are still growing rapidly, the filter may be transferred to an agar plate containing chloramphenicol (170-200 µg/ml) and incubated for 12 hours at 37°C. 13. Move the master nitrocellulose filter to a fresh LB (or SOB) agar plate containing the appropriate antibiotic and 25% glycerol. Then freeze it as described in Step 5. 14. Lyse the bacteria adhering to the replica filters and bind the liberated DNA to the nitrocellulose or nylon filter using the procedures described in Chapter 1, Protocol 31 . Proceed with hybridization as described in Chapter 1, Protocol 32 . REFERENCES 1. Hanahan D. and Meselson M. 1980. Plasmid screening at high colony density. Gene 10:63-67. printer friendly version Buy The Book | Our Vision | Take The Tour | Newsletter | Search CSHL Press Home | Contact | Members Home | CSHL Home Copyright © 2000. Cold Spring Harbor Laboratory Press. http://www.molecularcloning.com/members/protocol.jsp?pronumber=30&chpnumber=1 [2002-2-18 16:16:03]
@片Molecular Cloning printer friendy versio Protocol 31 Lysing Coloniesan Binding of DNA to Fiters atgeocoo0e6trHowe2a6g7awoagce5teee coN tra METHOD 1.pe()rand heon %SDS (o izing solution.Leave the of 3MM papor,which has boen saturated wth 2x SSPE y tide un on a theet ofdry 3MM panet and alo g for 1- 80a c32 ed DNAs that contain ●printer friendy versio g8ao
Chapter:1 Protocol:31 Lysing Colonies and Binding of DNA to Filters CHAPTER 1 > PROTOCOL 31 printer friendly version Protocol 31 Lysing Colonies and Binding of DNA to Filters In this protocol, based on the procedure of Grunstein and Hogness (1975), alkali is used to liberate DNA from bacterial colonies on nitrocellulose or nylon filters. The DNA is then fixed to the filter by UV-cross-linking or baking under vacuum. MATERIALS CAUTION: Please click for information about appropriate handling of materials. RECIPE: Please click for components of stock solutions, buffers, and reagents. Buffers and Solutions Denaturation solution Neutralizing solution SDS (10% w/v) 2X SSPE Vectors and Bacterial Strains E. coli transformants immoblized on filters Use transformants prepared by one of the methods described in Chapter 1, Protocol 28 , Chapter 1, Protocol 29 and Chapter 1, Protocol 30 . METHOD 1. Cut four pieces of Whatman 3MM paper (or an equivalent) to an appropriate size and shape and fit them neatly onto the bottoms of four glass or plastic trays. Saturate each of the pieces of 3MM paper with one of the following solutions: 10% SDS (optional) denaturizing solution neutralizing solution 2x SSPE 2. Pour off any excess liquid and roll a 10-ml pipette along the sheet to smooth out any air bubbles that occur between the 3MM paper and the bottom of the container. 3. Use blunt-ended forceps to peel the nitrocellulose or nylon filters from their plates and place them colony side up on the SDS-impregnated 3MM paper for 3 minutes. 4. After the first filter has been exposed to the SDS solution for 3 minutes, transfer it to the second sheet of 3MM paper saturated with denaturing solution. Transfer the remainder of the filters in the same order in which they were removed from their agar plates. Expose each filter to the denaturing solution for 5 minutes. 5. Transfer the filters to the third sheet of 3MM paper, which has been saturated with neutralizing solution. Leave the filters for 5 minutes. Optional: Repeat this step once. 6. Transfer the filters to the last sheet of 3MM paper, which has been saturated with 2x SSPE. Leave the filters for 5 minutes. 7. Dry the filters using one of the methods below. If the DNA is to be fixed to the filters by baking: Lay the filters, colony side up, on a sheet of dry 3MM paper and allow them to dry at room temperature for at least 30 minutes. If the DNA is to be fixed to the filters by cross-linking with UV light: Lay the filters on a sheet of 2x SSPE-impregnated 3MM paper or on dry paper, depending on the manufacturer's recommendation. 8. Fix the DNA to the filters using one of the methods below. For baking: Sandwich the filters between two sheets of dry 3MM paper, and fix the DNA to the filters by baking for 1-2 hours at 80°C in a vacuum oven. For cross-linking with UV light: Follow the manufacturer's instructions for fixing DNA to filters using a commercial device for this purpose. 9. Hybridize the DNA immobilized on the filters to a labeled probe as described in Chapter 1, Protocol 32 . REFERENCES 1. Grunstein M. and Hogness D.S. 1975. Colony hybridization: A method for the isolation of cloned DNAs that contain a specific gene. Proc. Natl. Acad. Sci. 72:3961-3965. printer friendly version Buy The Book | Our Vision | Take The Tour | Newsletter | Search CSHL Press Home | Contact | Members Home | CSHL Home Copyright © 2000. Cold Spring Harbor Laboratory Press. http://www.molecularcloning.com/members/protocol.jsp?pronumber=31&chpnumber=1 [2002-2-18 16:16:11]
Molecular Cloning CHAPTER1>PROTOCOL32 printer friendly ver Protocol 32 MATERIALS containing the METHOD he ep antrotang on s for 30 2 。 se b the ensure sscking ing them to x-tay 5im (k odak XAR-2 XAR-5 or thei on of the om the culture by one of the m REFERENCES
Chapter:1 Protocol:32 Hybridization of Bacterial DNA on Filters csh csh CHAPTER 1 > PROTOCOL 32 csh printer friendly version Protocol 32 Hybridization of Bacterial DNA on Filters This protocol describes procedures to hybridize DNA from transformed colonies immobilized on filters with radiolabeled probes and to recover from a master plate the corresponding colonies that hybridize specifically to the probe. The method is based on the procedure published by Grunstein and Hogness (1975). MATERIALS csh CAUTION: Please click for information about appropriate handling of materials. csh RECIPE: Please click for components of stock solutions, buffers, and reagents. Buffers and Solutions Formamide Prehybridization/hybridization solution (for Plaque/Colony Lifts) Prewashing solution (1-32) Wash solution 1 (1-32) Wash solution 2 (1-32) Wash solution 3 (1-32) Media Rich medium containing the appropriate antibiotic Nucleic Acids and Oligonucleotides Filters with immobilized DNA from transformed colonies Use filters prepared as described in Chapter 1, Protocol 31 . Probes 32P-labeled double-stranded DNA probe or Synthetic oligonucleotide probes Additional Reagents Step 15 of this protocol requires the reagents listed in Chapter 1, Protocol 1 or Chapter 1, Protocol 4 . Step 15 may also require the reagents listed in Chapter 8, Protocol 12 . METHOD 1. Float the baked or cross-linked filters on the surface of a tray of 2x SSC until they have become thoroughly wetted from beneath. Submerge the filters for 5 minutes. 2. Transfer the filters to a glass baking dish containing at least 200 ml of prewashing solution. Stack the filters on top of one another in the solution. Cover the dish with Saran Wrap and transfer it to a rotating platform in an incubator. Incubate the filters for 30 minutes at 50°C. 3. Gently scrape the bacterial debris from the surfaces of the filters using Kimwipes soaked in prewashing solution. This scraping ensures removal of colony debris and does not affect the intensity or sharpness of positive hybridization signals. 4. Transfer the filters to 150 ml of prehybridization solution in a glass baking dish. Incubate the filters with agitation for 1-2 hours or more at the appropriate temperature (i.e., 68°C when hybridization is to be carried out in aqueous solution; 42°C when hybridization is to be carried out in 50% formamide). 5. Denature 32P-labeled double-stranded DNA by heating to 100°C for 5 minutes. Chill the probe rapidly in ice water. Single-stranded probes need not be denatured. 6. Add the probe to the prehybridization solution covering the filters. Incubate at the appropriate temperature until 1-3 Cot1/2 is achieved. During the hybridization, keep the containers holding the filters tightly closed to prevent the loss of fluid by evaporation. Use between 2 x 105 and 1 x 106 cpm of 32P-labeled probe (specific activity approx. 5 x 107 cpm/µg) per milliliter of prehybridization solution. Using more probe will cause the background of nonspecific hybridization to increase, whereas using less will reduce the rate of hybridization. Hybridization mixtures containing radiolabeled single-stranded probes may be stored at 4°C for several days and reused without further treatment. In some cases, hybridization probes prepared from double-stranded DNA templates can be reused after freezing the solution, thawing, and boiling for 5 minutes in a chemical fume hood. 7. When the hybridization is complete, remove the hybridization solution and immediately immerse the filters in a large volume (300-500 ml) of Wash solution 1 at room temperature. Agitate the filters gently and turn them over at least once during washing. After 5 minutes, transfer the filters to a fresh batch of wash solution and continue to agitate them gently. Repeat the washing procedure twice more. 8. Wash the filters twice for 0.5-1.5 hours in 300-500 ml of Wash solution 2 at 68°C. 9. Dry the filters in the air at room temperature on 3MM paper. Streak the underside of the filters with a water-soluble glue stick and arrange the filters (numbered side up) on a clean, dry, flat sheet of 3MM paper. Press the filters firmly against the 3MM paper to ensure sticking. 10. Apply adhesive dot labels marked with either radioactive ink or chemiluminescent markers to several asymmetric locations on the 3MM paper. Cover the filters and labels with Saran Wrap. Use tape to secure the wrap to the back of the 3MM paper and stretch the wrap over the paper to remove wrinkles. 11. Analyze the filters by phosphorimaging or exposing them to X-ray film (Kodak XAR-2, XAR-5, or their equivalents) for 12-16 hours at -70°C with an intensifying screen. 12. Develop the film and align it with the filters using the marks left by the radioactive ink. Use a nonradioactive fiber-tip pen in a nonblack color to mark the film with the positions of the asymmetrically located dots on the numbered filters. 13. Tape a piece of clear Mylar or other firm transparent sheet to the film. Mark on the clear sheet the positions of positive hybridization signals. Also mark (in a different color) the positions of the asymmetrically located dots. Remove the clear sheet from the film. Identify the positive colonies by aligning the dots on the clear sheet with those on the agar plate. 14. Use a sterile toothpick or inoculating needle to transfer each positive bacterial colony into 1-2 ml of rich medium (e.g., LB, YT, or Terrific Broth) containing the appropriate antibiotic. 15. After a period of growth, plasmid DNA can be isolated from the culture by one of the minipreparation methods described in Chapter 1, Protocol 1 and Chapter 1, Protocol 4 and can be further analyzed by restriction endonuclease digestion or by PCR. REFERENCES 1. Grunstein M. and Hogness D.S. 1975. Colony hybridization: A method for the isolation of cloned DNAs that contain a specific gene. Proc. Natl. Acad. Sci. 72:3961-3965. http://www.molecularcloning.com/members/protocol.jsp?pronumber=32&chpnumber=1 (1 / 2) [2002-2-18 16:17:44]