Manual: Lambda DASH Library
Contents
1. A 5 DNA Screening Protocol AP vai ostaen oastei vistes 6 Prehy bridization lana iria lalla 7 HN DIIdIZA OM ci A ed Le least weil ins be ee eee te 8 Hybridization SOM OD citi cie amp WASHES 9 Exposure to cai sins an nahi ease ee an gies 10 Secondary s cccccscccrcdcecssesvsssossaceseascsvensosdasccevascesassesssesesscsecasensdsesuss sdesescoenssdesasssoasoosssssessed es 10 Tertiary A A svesessesstuadstsesossaesdcccsetenssesetesssssscsanaeses 10 Phage NA O 11 Making End Specific RNA Probes Using the Lambda DASH Vector cocnconnconnnonnnonnnconocnnconnconnoss 11 Transcription risi ise a RAGS eis io 12 Rapid Restriction Mapping erica iure lune soe erat 12 Preparation of Media and ReagentSs verrrrrrrrrrereree nere ce nere se nre neneeee nese se nre rene resenenee nese seenesenenecneneo 13 Reference 14 AA NO 14 MSDS IOMA 14 Lambda DASH Library MATERIALS PROVIDED Material provided Quantity Amplified premade library constructed in the Lambda DASH vector 1 ml Host strains XL1 Blue MRA strain 0 5 ml bacterial glycerol stock XL1 Blue MRA P2 strain 0 5 ml bacterial glycerol stock Premade libraries have been amplified one time and frozen in the presence of 7 DMSO Upon arrival store at 80 C2. modified to enhance the stability of clones containing methylated DNA in addition they enhance the stability of nonstandard DNA structures Media for bacterial Bacterial Plates for Media for cultures for titering phage strain bacterial streak glycerol stock final concentration XL1 Blue LB LB LB with 0 2 v v maltose MRA P2 10 mM MgSO XL1 Blue LB LB LB with 0 2 v v maltose MRA 10 mM MgSO On arrival prepare the following from the glycerol stock Note not allow the contents of the vial to thaw The vials can be stored at 20 C or 80 but most strains remain viable longer if stored at 80 C Revive the stored cells by scraping off splinters of solid ice with a sterile wire loop Streak the splinters onto an LB plate containing the appropriate antibiotic Restreak the cells fresh each week Preparation of a 80 C Glycerol Stock 1 3 In a sterile 50 ml conical inoculate 10 ml of appropriate liquid media with one or two colonies from the plate Grow the cells to late log phase Add 4 5 ml of a sterile glycerol liquid media solution 5 ml of glycerol 5 ml of appropriate media to the bacterial culture from step 1 Mix well Aliquot into sterile centrifuge tubes 1 ml tube This preparation may be stored at 20 C for 1 2 years or at 80 for more than 2 years See Preparation of Media and Reagents Lambda DASH Library PREPARATION OF
3. Store the plates at 4 C overnight with gentle rocking if possible This allows the phage to diffuse into the buffer 8 Recover the bacteriophage suspension from each plate using a 10 ml disposable pipette and pool it into a sterile polypropylene container Rinse the plates with an additional 2 ml of SM buffer and pool Add chloroform to a 5 final concentration Mix well and incubate for 15 minutes at room temperature 9 Remove the cell debris by centrifugation for 10 minutes at 2000 x g Lambda DASH Library 10 Recover the supernatant and transfer to a sterile polypropylene or glass bottle Add chloroform to a 0 3 final concentration and store at 4 C We recommend storing aliquots in 7 dimethylsulfoxide DMSO at 80 C 11 Check the titer of the amplified library using host cells and serial dilutions of the library Assume 10 10 pfu ml DNA SCREENING PROTOCOL 1 Titer the library to determine the concentration prepare fresh host cells to use in titering and in screening 2 Plate on large 150 mm NZY plates 2 or more days old to 50 000 pfu plate with 600 ul of ODgo 0 5 host cells plate and 6 5 ml of NZY top agar plate use 20 plates to screen 1 x 10 pfu 3 Incubate at 37 C for 8 hours or if plating late in the day grow overnight 4 Refrigerate the plates for 2 hours at 4 C to chill This helps prevent top agar from sticking to the nitrocellulose filter Note Use forceps and wear gloves for
4. PLATING CULTURES Day 1 1 2 Day 2 3 4 5 TITERING PROCEDURE Inoculate 50 ml of LB broth supplemented with 0 2 maltose and 10 mM MgSO in a sterile flask with a single colony of the appropriate bacterial host Note DO NOT add antibiotic to the overnight culture or to the titering plates Grow overnight with shaking at 30 C This temperature ensures that the cells will not overgrow Phage can adhere to dead cells as well as to live ones and can lower the titer Spin the cells down in a sterile conical tube for 10 minutes at 2000 rpm Carefully decant the media off the cell pellet and gently resuspend the pellet in 15 ml of 10 mM MgSO Do not vortex Dilute the cells to ODgoo 0 5 with 10 mM MgSO Approximately 600 ul of 0 5 cells are needed for each 150 mm plate and 200 ul of 0 5 cells for each 100 mm plate The cells may be stored for 2 3 days at 4 C Prepare the host bacteria as outlined in Preparation of Plating Cultures Make dilutions of the lambda phage in SMS buffer Add 1 pl of the lambda phage to 200 pl of host cells diluted in 10 mM MgSO to 0 5 If desired also add 1 ul of a 1 10 dilution of the lambda phage in SM buffer to 200 ul of host cells Incubate the phage and bacteria for 15 minutes at 37 C to allow the phage to attach to the cells Best results are obtained with gentle shaking Add 2 5 3 ml of NZY top agar 48 C and plate on NZY plates Count the
5. plaques and determine the plaque forming units per milliliter pfu ml concentration of the library See Preparation of Media and Reagents Lambda DASH Library AMPLIFICATION PROCEDURE OPTIONAL Day 1 Day 2 Day 3 Note The premade library has been through one round of amplification It is usually desirable to amplify libraries prepared in lambda vectors to make a large stable quantity of a high titer stock of the library However more than one round of amplification is not recommended since slower growing clones may be significantly underrepresented 1 Prepare the host bacteria as outlined in Preparation of Plating Cultures 2 Dilute the cells to OD6o 0 5 in 10 mM MgSO 600 ul of OD 699 0 5 cells are needed per 150 mm plate 3 aliquots of the diluted phage containing 50 000 plaque forming units with 600 ul of 0 5 host cells in 14 ml BD Falcon polypropylene tubes Note not add more than 300 ul of phage volume per 600 ul of cells 4 Incubate the tubes containing the phage and host cells for 15 minutes at 37 C 5 Mix 6 5 ml of melted NZY top agar with each aliquot of infected bacteria and spread evenly onto a freshly poured 150 mm NZY plate of bottom agar make sure the top agar is cooled to 48 C before adding it to the aliquot 6 Incubate the plates at 37 C for 6 8 hours Do not allow the plaques to get larger than 1 2 mm 7 Overlay the plates with 8 10 ml of SM buffer
6. primer labeling kit Catalog 300385 which is designed to produce high specific activity DNA probes in 2 minutes Double stranded probes can also be nick translated with fresh a 32P dATP It is best to use 1 106 5 106 counts ml of hybridization solution Keep the concentration of counts high and use 1 107 counts filter Hybridization Solution For Oligonucleotides 6x SSC buffer 20 mM NaH PO 0 4 SDS Denatured sonicated salmon sperm DNA Prepare the hybridization solution and warm the solution to the appropriate temperature Boil the salmon sperm DNA and then add it to the prewarmed hybridization solution Pour out the prehybridization solution buffer from the hybridization bag Add the hybridization solution and then the appropriate amount of labeled oligonucleotide See Preparation of Media and Reagents For Stratagene s Duralon UV membranes increase the SDS concentration to 1 w v Lambda DASH Library 4 Heat seal and hybridize at 5 10 C below the melting temperature 7 Calculate the 7 using the following formula Note The first method below overestimates the of hybrids involving longer nucleotides The second formula works only for Na concentrations of lt 1 M Oligonucleotides Shorter than 18 Bases Tm 2 C A 4 C G Oligonucleotides 14 Bases and Longer up to 60 70 Nucleotides Tm 81 5 16 6 log o Na 0 41 G 600 N where N c
7. the following steps 5 Transfer for 2 minutes onto nitrocellulose filters prick the filter with a needle through the membrane into the agar for orientation If desired you may use waterproof ink in a syringe needle Note If making duplicates allow the second filter to transfer for 4 minutes Pyrex dishes are convenient All solutions should be at room temperature a Denature the filter after lifting by submerging the filter in 1 5 M NaCl and 0 5 M NaOH for 2 minutes Note fusing charged nylon wash with gloved fingertips to remove the excess top agar b Neutralize for 5 minutes by submerging the filter in 1 5 M NaCl and 0 5 Tris HCl pH 8 0 Rinse for 30 seconds only submerged in 0 2 Tris HCl pH 7 5 and 2 SSC Blot briefly on Whatman 3MM paper See Preparation of Media and Reagents 6 Lambda DASH Library Prehybridization 7 Either UV crosslink the DNA to the filters or bake at 80 C for 1 5 2 hours 8 Store the agar stock plates of the transfers at 4 C to use after screening Prehybridization for Oligo Probe 6x SSC 20 mM NaH PO 0 4 sodium dodecyl sulfate SDS 5 Denhardt s Denatured sonicated salmon sperm DNA OR Prehybridization for Double Stranded Probe 2 Pipes buffer 50 Deionized formamide 0 5 SDS Denatured sonicated salmon sperm DNA The amount of solution to make is dependent on the number of filters used generally 2 3 ml 137 mm membran
8. 00800 7001 7001 0800 023 0448 00800 7400 7400 0800 563 080 0800 563 082 0800 563 081 00800 7000 7000 00800 7001 7001 00800 7400 7400 0800 917 3282 0800 917 3283 0800 917 3281 All Other Countries Please contact your local distributor A complete list of distributors is available at www stratagene com Lambda DASH Library CONTENTS Materials Provided cccscccssscssscssscssscnssenssensccessssssscsscssscssssnsensssnessesssessssssssssssssssssescoescoessnessoes 1 Storage AAA NN 1 Additional Materials Required csscccssssccssssseecscsccsscscssccssscceecsecsesccsesceesescesssessesccsseseeesseseesees 1 Host Strains and Genotypes ccccsscsccscssccecssscesccccsscccsssseecssccesceccescessssceesscsssscccsescessssseesessesseseesees 1 Introduction 2 Preparation of H st Cells ccscisssscssceccesseactovessesesssscastevsassesdesbecassoenstonsssbstessosesseracceseasesbesssessoceassensasesess 3 Preparation of 80 Glycerol Stock sass sola cise a 3 Preparation of Plating Cultures sssssscssssscccsssscsscssccecssscesscccesccesssceecscssssccsesceesessesssessesessesees 4 O iaia lia illo 4 Day Trudi ee Aes Ga nL AI Rea aA ae a ehe 4 Mat sScvasesabsdcesassubses sosocepsscsesescvents 4 Amplification Procedure optional ccrrrrerrrrrerrce sese senese ererenenecee nese ne 5 DI li dia 5 Day ZA A ee A 5 A E A AA
9. DA DASH VECTOR T3 and T7 promotors flanking the insertion sites of the Lambda DASH vector permit the generation of end specific hybridization probes Once a recombinant clone containing an insert is isolated it is simple to make end specific probes 1 Grow a small preparation of the phage DNA using a liquid mini prep protocol Do not use plate lysates because the agar contains restriction enzyme inhibitors Digest several micrograms of the DNA to completion with Rsa I or Pall These enzymes should cleave the insert close to the RNA polymerase promotors on average 256 nucleotides from promotors Add a 1 10 volume of 10x STE Add proteinase K to a final concentration of 200 ug ml and incubate at 37 C for 30 minutes Extract twice with phenol chloroform 1 1 v v Ethanol precipitate and dry the pellet Resuspend the pellet in 10 ul of TE buffer See Preparation of Media and Reagents Lambda DASH Library Transcription Rapid Restriction The following is a typical reaction for generating high specific activity probes Add the solutions in the following order 5 ul of 5x transcription buffers 1 ug of restricted DNA template 1 ul of 10 mM rATP 1 ul of 10 mM rGTP 1 ul of 10 mM rCTP 1 ul of 0 75 M dithiothreitol DTT 5 ul of 400 800 Ci mmol 10 uCi pl 032P rUTP 10 U of T3 or T7 RNA polymerase Diethylpyrocarbonate DEPC treated water to a final volume of 25 ul Incubate at 37 C for 30 minut
10. Do not pass through more than two freeze thaw cycles STORAGE CONDITIONS Premade Library 80 C Bacterial Glycerol Stocks 80 C ADDITIONAL MATERIALS REQUIRED 14 ml BD Falcon polypropylene round bottom tubes BD Biosciences Catalog 352059 Sonicated salmon sperm DNA 100 ug ml Stratagene Catalog 201190 NucTrap probe purification columns Stratagene Catalog 400701 25 columns and 400702 50 columns HOST STRAINS AND GENOTYPES Host Strains Genotypes XL1 Blue MRA A mcrA 183 A merCB hsdSMR mrr 1 73 endAl supE44 thi 1 gyrA96 relA1 lac XL1 Blue MRA P2 XL1 Blue MRA P2 lysogen Revision A Agilent Technologies Inc 2009 Lambda DASH Library 1 INTRODUCTION Left End 0 Bgl Il 0 42 The Lambda DASH vector is a versatile genomic replacement lambda phage vector designed to allow high resolution restriction mapping and rapid chromosomal walking without subcloning The vector accepts inserts from 9 to 23 kb The Lambda DASH system takes advantage of spi sensitive to P2 inhibition selection Lambda phages containing active red and gam genes are unable to grow on host strains that contain P2 phage lysogens Lambda phages without these genes are able to grow on strains lysogenic for P2 such as XL1 Blue MRA P2 a P2 lysogen of XL1 Blue MRA The red and gam genes in the Lambda DASH DNA are located on the stuffer fragment therefore the wild type Lambda DASH phage cannot grow on X
11. L1 Blue MRA P2 When the stuffer fragment is replaced by an insert the recombinant Lambda DASH DNA becomes red gam and the phage is able to grow on the P2 lysogenic strain Therefore by plating the library on the XL1 Blue MRA P2 strain only recombinant phages are allowed to grow The strain XL1 Blue MRA is also provided as a control strain and later for growth of the recombinant when the selection is no longer necessary Digestion with Sa I allows excision of the insert and flanking T3 and T7 bacteriophage promotors as an intact cassette T3 and T7 promotors flanking the insertion sites can be used to generate end specific RNA probes for use in chromosomal walking and restriction mapping 24 80 25 30 126 16 129 17 Xba 32 77 Xho 32 77 Sac 32 78 1 29 03 Hind 111 20 03 Sac 20 03 Xho 20 03 Xba 20 04 Hind 111 32 78 BamH 32 78 Ay EcoR 32 79 Xba 32 81 Bgl Il 34 02 Bgl Il 34 33 Bgl Il 34 98 Xba 19 99 BamH 20 02 Bal Il 35 04 Sal 20 00 Kpn 17 05 Kpn 18 56 EcoR 20 02 N Sal 32 81 Right End 41 90 Sal Sa Bg Bg Bg J 3535 ninL44 lt KH54 nin5 FIGURE 1 Map of the Lambda DASH replacement vector Lambda DASH Library PREPARATION OF HOST CELLS The host strains have been sent as glycerol stocks For the appropriate media and plates please refer to the following table Note The strains XL1 Blue MRA and XL1 Blue MRA P2 have been
12. Lambda DASH Library INSTRUCTION MANUAL Revision A BN 943801 12 For In Vitro Use Only 943801 12 LIMITED PRODUCT WARRANTY This warranty limits our liability to replacement of this product No other warranties of any kind express or implied including without limitation implied warranties of merchantability or fitness for a particular purpose are provided by Agilent Agilent shall have no liability for any direct indirect consequential or incidental damages arising out of the use the results of use or the inability to use this product ORDERING INFORMATION AND TECHNICAL SERVICES United States and Canada Agilent Technologies Stratagene Products Division 11011 North Torrey Pines Road La Jolla CA 92037 Telephone Order Toll Free Technical Services Internet World Wide Web 858 373 6300 800 424 5444 800 894 1304 techservices agilent com www stratagene com Europe Location Telephone Fax Technical Services Austria 0800 292 499 0800 292 496 0800 292 498 Belgium 00800 7000 7000 00800 7001 7001 00800 7400 7400 0800 15775 0800 15740 0800 15720 France 00800 7000 7000 00800 7001 7001 00800 7400 7400 0800 919 288 0800 919 287 0800 919 289 Germany 00800 7000 7000 00800 7001 7001 00800 7400 7400 0800 182 8232 0800 182 8231 0800 182 8234 Netherlands Switzerland United Kingdom 00800 7000 7000 00800 7001 7001 00800 7400 7400 0800 023 0446 00800 7000 7000 31 0 20 312 5700
13. e 1 Preheat the prehybridization solution to 50 C without the salmon sperm DNA Preboil the salmon sperm DNA 10 minutes and add it to the warm prehybridization solution 2 Wet each filter quickly in the prehybridization buffer in a tray placing each filter on top of the next until each is wet through Add more prehybridization solution as necessary This helps wet the filters completely to allow more even hybridization later 3 Put the wet prehybridization filter stack in a heat seal bag add the remaining prehybridization buffer and heat seal 4 Calculate the hybridization temperature generally 42 C and prehybridize for a minimum of 2 hours 5 Prehybridize and hybridize a blank nitrocellulose filter background along with the rest and wash it to determine when and at what temperature the background counts disappear For Stratagene s Duralon UV membranes increase the SDS concentration to 1 See Preparation of Media and Reagents Lambda DASH Library Hybridization Labeling Oligonucleotide Probes Label oligonucleotides with fresh y 2P ATP High specific activity y yields the best results 1 Perform a polynucleotide kinase labeling in 1 ligase buffer for 30 minutes at 37 C Incubate for 15 minutes at 65 C to inactivate the kinase Run the solution over a G 50 column to get rid of the unincorporated counts Labeling Double Stranded Probes Stratagene offers the Prime It random
14. e activity and the addition of cold rUTP has been shown to enhance polymerase activity especially for T7 transcriptions See Preparation of Media and Reagents Lambda DASH Library PREPARATION OF MEDIA AND REAGENTS Note All media must be autoclaved before use LB Broth per Liter 10 g of NaCl 10 g of bactotryptone 5 g of yeast extract NZY Broth per Liter 5 g NaCl 2 g MgSO 7H 0 5 g Yeast extract 10 g NZ Amine casein hydrolysate Adjust to pH 7 5 with NaOH LB Plates per Liter NZY Top Agar per 100 ml LB broth NZY broth 15 g of Difco agar 0 7 w v agarose 20x SSC NZY Plates per Liter 175 3 g NaCl NZY broth 88 2 g Sodium citrate 15 g of Difco agar 800 0 ml Water Adjust to pH 7 0 with a few drops of a 10 0 N NaOH Adjust volume to 1 liter with water 10x STE SM Buffer per Liter 1M NaCl 5 8 g of NaCl 200 mM Tris HCl pH 7 5 2 0 g of MgSO H O 100 mM EDTA 50 0 ml of Tris HCl pH 7 5 5 0 ml of 2 w v gelatin Adjust volume to 1 liter with water 10x Pipes 10x Ligase Buffer 4 0 M NaCl 500 mM Tris HCl pH 7 5 0 1 M Pipes pH 6 5 70 mM MgCl 10 mM DTT 5x Transcription Buffer 200 mM Tris HCl pH 8 0 40 mM MgCl 10 mM spermidine 250 mM NaCl Lambda DASH Library REFERENCE 1 Sambrook J Fritsch E F and Maniatis T 1989 Molecular Cloning A Laboratory Manual Cold Spring Harbor Laboratory Press Cold Spring Harbor NY ENDNOTES Whatman is a reg
15. es If desired unincorporated nucleotides may be removed over a 1 ml Sephadex G 50 column however ribonuclease may be present in the column and could degrade the probe It is recommended to examine a small aliquot of the probe 3 ul on a 5 acrylamide 7 M urea gel with single stranded radioactive size markers to determine the size of the probe Probes less than 50 nucleotides in length will not be useful because they do not hybridize well Probes greater than 1000 nucleotides in length run a higher risk of containing a highly repeated sequence Because not all enzymes produce ideal probes it is best to digest the DNA with several four base restriction enzymes and test the probes in parallel 4 u I can be used if Pal I and Rsa I are not suitable The probe is now ready to use for hybridization These probes can be used to rescreen the library to isolate overlapping clones Stratagene offers the NucTrap probe purification columns Stratagene Catalog 400701 25 columns and 400702 50 columns a rapid and efficient way to purify probes away from unincorporated nucleotides Mapping Digestion with Sa I will excise the insert DNA with the T3 and T7 promoter sequences as an intact fragment from the Lambda DASH arms Optional Add 40 U of RNase Block Ribonuclease Inhibitor Stratagene Catalog 300151 4000 U and 300152 16 000 U and 1 ul of 10 mM rUTP to the transcription reaction RNase Block Ribonuclease Inhibitor inhibits any RNas
16. hain length For Double Stranded Probes 2 Pipes buffer 50 Deionized formamide 0 5 SDS Denatured sonicated salmon sperm DNA 1 Prepare the solution 2 Warm the solution to 42 C boil the appropriate amount of salmon sperm with the probe for 4 minutes and then add it to the hybridization buffer 3 Decant the prehybridization buffer and replace it with the hybridization solution and probe 4 Hybridize overnight at 42 C Washes Oligo Probes Use 6x SSC 0 1 SDS at 10 C If the probe sequence is unknown start with a room temperature wash and gradually increase the temperature until the background diminishes DO NOT allow the membranes to completely dry out or the probe may be irreversibly bound Double Stranded Probes Use 0 1 SSC and 0 1 SDS and wash the filters at 50 65 C with shaking For Stratagene s Duralon UV membranes increase the SDS concentration to 1 Lambda DASH Library 2 Exposure to Film After washing remove the excess liquid by blotting on Whatman 3 MM paper and place between two sheets of plastic wrap in a cassette with intensifying screens Expose overnight at 80 C If the filters are kept slightly moist between plastic wrap a high background can be washed again Expose and develop the film SECONDARY SCREENING TERTIARY SCREENING To orient the filters line up the film and mark numbers and dots where the needle poked through Determine the putative clone
17. istered trademark of Whatman Ltd MSDS INFORMATION The Material Safety Data Sheet MSDS information for Stratagene products is provided on the web at http www stratagene com MSDS Simply enter the catalog number to retrieve any associated MSDS s in a print ready format MSDS documents are not included with product shipments 14 Lambda DASH Library
18. s with the strongest signal on film Cut out a square centimeter window from the stock plate where the putative clone lines up with the film spot Use a Pasteur pipet a scalpel or an inverted yellow tip Put into 1 ml of SM buffer and 20 ul of chloroform Vortex Dilute and titer with host cells on a 100 mm NZY plate so that one plate will have 50 plaques and the second plate will have 500 plaques Incubate overnight at 37 C Make plaque lifts from prechilled plates and perform prehybridization and hybridization as before Isolated plaques may be picked from a secondary screen If the positive plaques are too close to the background plaques then core retiter and perform a tertiary screen to obtain the isolates A cored plaque in 1 ml of SM buffer represents 1 x 106 pfu Line up the filter with the film as before to cut out the plaque These cored candidate clones are stored at 4 C in 1 ml of SM buffer with 20 ul of chloroform Remember that phage diffuses in agar so the screening procedure should be done quickly after lifts are taken Once the plaque isolate has been cored from the plate and put in SM buffer with a drop of chloroform it is stable at 4 C for a few months We recommend growing a high titer stock and storing the phage at 80 C in SM buffer and 7 DMSO Lambda DASH Library PHAGE PREPARATIONS Refer to reference for large scale phage preparations MAKING END SPECIFIC RNA PROBES USING THE LAMB
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