ABLE C Strain ABLE K Strain Excision Protocol Manual
Contents
1. amplified lambda phage should be excised than is found in the primary library to ensure statistical representation of the excised clones The ExAssist helper phage should be added at a 1 1 phage to cell ratio For example use 5 ml of ODs 5 0 cells 10 of the ExAssist helper phage 10 pfu of the amplified library Incubate the conical tube at 37 C for 15 minutes Add 3 ml of LB broth 25 ml of LB broth for mass excision and incubate the conical tube for 2 212 hours at 37 C with shaking Incubation times for mass excision in excess of 3 hours may alter the clonal representation Single clone excision reactions can be safely performed overnight since clonal representation is not relevant Note Cloudy growth may not always be seen ABLE C Strain and ABLE K Strain Excision Protocol 10 Heat the conical tube at 70 C for 15 minutes and then spin the tube again at 4000 x g for 15 minutes 11 Decant the supernatant into a sterile conical tube This stock contains the excised pBluescript phagemid packaged as filamentous phage particles The stock may be stored at 4 C for 1 2 months 12 To plate the excised phagemids add 200 ul of freshly grown SOLR cells from step 6 OD 1 0 to two 1 5 ml microcentrifuge tubes Add 100 ul of the phage supernatant 1 ul of the phage supernatant for mass excision from step 11 above to one microcentrifuge tube and 10 pl of the phage supernatant to the other microcentrifuge tube 132. ABLE C Strain and ABLE K Strain Excision Protocol Instruction Manual Catalog 200305 ABLE C strain and ABLEK strain 200306 ABLE C strain 200307 ABLEK strain Revision B Research Use Only Not for Use in Diagnostic Procedures 200305 12 OX Agilent Technologies 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 Email techservices agilent com World Wide Web www genomics agilent com Telephone All Other Countries Please visit www genomics agilent com and click Contact Us ABLE C Strain and ABLE K Strain Excision Protocol CONTENTS Materials ucnuD Ci Sone es s Sne Host Strains and Genotypes 5 esiti edi recede de sheets lees teer re occae ce dees Stor ge Conditions tH M as Additional Materials Required for the Excision Protocol sesserseesoeseeseesoeseesoesossoecoesseseorsessoesee Tntrod ction sessioon E esiste a a Ure aie ABLE C Strain and ABLE K Strain Excision Protocol MATERIALS PROVIDED
3. Host Strains and Genotypes Host strain Reference Genotype ABLE C strain E coli C lac LacZo Kan McrA McrCB McrF Mrr HsdR r m F proAB lacl ZAM15 Tn10 Tet ABLE K strain E coli C lac LacZo Kan McrA McrCB McrF Mrr HsdR ry m F proAB lacPZAMT5 Tn10 Tet a For host strain shipping and storage conditions please see Preparation of Host Cells STORAGE CONDITIONS Bacterial Glycerol Stocks 80 C ADDITIONAL MATERIALS REQUIRED FOR THE EXCISION PROTOCOL f1 Helper Phage ExAssist interference resistant helper phage gt 1 x 10 pfu ml Host strains SOLR strain and XL1 Blue strain see table below for genotypes Additional Host Strain Genotypes Hoststrim Genotype S SOLR strain el4 McrA A mcrCB hsdSMR mrr 171 sbcC recB recJ uvrC umuC Tn5 Kan lac gyrA96 relAl thi 1 endAl A F proAB lacZAM15 Su nonsuppressing XL1 Blue strain recAl endAl gyrA96 thi 1 hsdR17 supE44 relA1 lac F proAB lacPZAM15 Tn10 Tet The SOLR strain should be used only for excision Revision B O Agilent Technologies Inc 2010 ABLE C Strain and ABLE K Strain Excision Protocol INTRODUCTION During cDNA library screening in plasmid systems many clones cannot be propagated because their products are lethal to the host Likewise while toxic cDNA clones can be propagated in lambda systems problems arise when attempting to subclone into plasmid systems which require survival of
4. Repeat step 12 using both the ABLE C and the ABLE K strains 14 Incubate the microcentrifuge tubes at 37 C for 15 minutes 15 Plate 100 ul from each microcentrifuge tube on LB ampicillin agar plates 50 ug ml see Media and Reagents and incubate the plates overnight at 37 C Due to the high efficiency of the excision process it may be necessary to titrate the supernatant to achieve single colony isolation Colonies appearing on the plate contain the pBluescript double stranded phagemid with the cloned DNA insert Helper phage will not grow since helper phage is unable to replicate in Su nonsuppressing SOLR ABLE C and ABLE K strains SOLR cells are also resistant to lambda phage infection thus preventing lambda phage contamination after excision To maintain the pBluescript phagemid streak the colony on a new LB ampicillin agar plate For long term storage prepare a bacterial glycerol stock and store at 80 C see Preparation of a 80 C Bacterial Glycerol Stock TROUBLESHOOTING Observation Low colony number Suggestion Excision efficiencies are directly related to the phage titer If an excision is unsuccessful it may be necessary to make a high titer stock of the phage and to repeat the excision procedure Incubate the plaque cores in SM buffer overnight to elute phage completely ABLE C Strain and ABLE K Strain Excision Protocol 5 PREPARATION OF MEDIA AND REAGENTS SM Buffer per L
5. elper phage with SOLR strain is designed to allow the efficient excision of the pBluescript phagemid from Lambda ZAP vectors while preventing problems associated with helper phage co infection The ExAssist helper phage contains an amber mutation that prevents replication of the phage genome in a nonsuppressing E coli strain such as SOLR or ABLE cells This allows only the excised phagemid to replicate in the host removing the possibility of productive co infection from the ExAssist helper phage Since the ExAssist helper phage cannot replicate in the SOLR or ABLE strains single stranded rescue cannot be performed in these strains using this helper phage Note The ExAssist interference resistant helper phage has a complementing D galactosidase sequences which may interfere with sequencing or site directed mutagenesis where oligonucleotide primers hybridize to B galactosidase sequences e g M13 20 primer The size of the ExAssist helper phage DNA single strand is 5 kb Mass excision can be used to generate subtraction libraries and subtraction DNA probes Converting the library to the phagemid form also allows screening of the phage library in eukaryotic cells by transformation of eukaryotic cells with supercoiled plasmid DNA 1 Core the plaque of interest from the agar plate and transfer the plaque to a sterile microcentrifuge tube containing 500 ul of SM buffer see Media and Reagents and 20 wl of chloroform Vortex the microce
6. iter 5 8 g of NaCl 2 0 g of MgSO 7H O 50 0 ml of 1 M Tris HCI pH 7 5 5 0 ml of 296 w v gelatin Add H50 to a final volume of 1 liter Autoclave LB Agar per Liter 10 g of NaCl 10 g of tryptone 5 g of yeast extract 20 g of agar Adjust pH to 7 0 with 5 N NaOH Add deionized H O to a final volume of 1 liter Autoclave Pour into petri dishes 225 ml 100 mm plate LB Broth per Liter 10 g of NaCl 10 g of tryptone 5 g of yeast extract Adjust to pH 7 0 with 5 N NaOH Add deionized H O to a final volume of 1 liter Autoclave LB Ampicillin Agar per Liter 1 liter of LB agar Autoclave Cool to 55 C Add 50 mg of filter sterilized ampicillin Pour into petri dishes 25 ml 100 mm plate LB Tetracycline Broth per Liter 1 liter of LB broth Autoclave Cool to 55 C Add 12 5 mg of filter sterilized tetracycline Store broth in a dark cool place as tetracycline is light sensitive LB Tetracycline Agar per Liter 1 liter of LB agar Autoclave Cool to 55 C Add 12 5 mg of filter sterilized tetracycline Pour into petri dishes 25 ml 100 mm plate Store plates in a dark cool place or cover plates with foil if left out at room temperature for extended time periods as tetracycline is light sensitive LB Tetracycline Kanamycin Broth per Liter 1 liter of LB broth Autoclave Cool to 55 C Add 12 5 mg of filter sterilized tetracycline Add 50 mg of filter sterilized kanamycin Store broth in a dark c
7. not required ABLE K strain LB kanamycin tetracycline LB kanamycin tetracycline LB supplement not required See Preparation of Media and Reagents 50 ug ml 12 5 pg ml On arrival prepare the following from the bacterial glycerol stock Note The host strains may thaw during shipment The vials should be stored immediately at 20 or 80 C but most strains remain viable longer if stored at 80 C It is also best to avoid repeated thawing of the host strains in order to maintain extended viability 1 Revive the stored cells by scraping off splinters of solid ice with a sterile wire loop 2 Streak the splinters onto an LB agar plate see Media and Reagents containing the appropriate antibiotic Restreak the cells fresh each week ABLE C Strain and ABLE K Strain Excision Protocol Preparation of a 80 C Bacterial Glycerol Stock 1 In a sterile 50 ml conical tube inoculate 10 ml of the appropriate liquid media with one or two colonies from the plate Grow the cells to late log phase 2 Add 4 5 ml of a sterile glycerol liquid media solution prepared by mixing 5 ml of glycerol 5 ml of liquid media to the bacterial culture from step 1 Mix well 3 Aliquot into sterile centrifuge tubes 1 ml tube This preparation may be stored at 20 C for 1 2 years or at 80 C for more than 2 years In Vivo EXCISION PROTOCOL USING EXAsSIST HELPER PHAGE Day 1 The ExAssist h
8. ntrifuge tube to release the phage particles into the SM buffer Incubate the microcentrifuge tube for 1 2 hours at room temperature or overnight at 4 C This phage stock is stable for up to 1 year at 4 C ABLE C Strain and ABLE K Strain Excision Protocol 3 Day 2 Grow an overnight culture of XL1 Blue cells in LB broth supplemented with 0 2 w v maltose 10 mM MgSO at 30 C In addition grow a 5 ml overnight culture of SOLR ABLE C and ABLE K cells in LB broth containing 12 5 ug ml of tetracycline and 50 ug ml of kanamycin at 30 C Make a 1 100 dilution of the cells using 0 5 ml of the overnight cultures and 50 ml of LB broth Grow this culture at 37 C for 23 hours to mid log phase ODgo 0 2 0 5 Gently spin down the XL1 Blue cells at 1500 x g Resuspend the cells at an ODgoo of 1 0 for single clone excision and at an ODqq of 5 0 in 10 mM MgSO for mass excision Allow the SOLR ABLE C and ABLE K cells to grow to an ODgo of 0 5 1 0 while continuing with steps 7 11 Before the SOLR ABLE C and ABLE K cells reach an OD 1 remove the cells from the 37 C incubator to room temperature Note Use these cells on the same day Combine the following components in a 50 ml conical tube 200 ul of XL1 Blue cells at an OD of 1 0 250 ul of phage stock containing gt 1 x 10 phage particles 1 pl of ExAssist helper phage 21 x 10 pfu ul Note When excising an entire library 2100 fold more of the
9. ool place as tetracycline is light sensitive LB Tetracycline Kanamycin Agar per Liter 1 liter of LB agar Autoclave Cool to 55 C Add 12 5 mg of filter sterilized tetracycline Add 50 mg of filter sterilized kanamycin Pour into petri dishes 25 ml 100 mm plate Store plates in a dark cool place or cover plates with foil if left out at room temperature for extended time periods as tetracycline is light sensitive ABLE C Strain and ABLE K Strain Excision Protocol REFERENCES 1 Greener A 1993 Strategies 6 1 7 9 Short J M and Sorge J A 1992 Methods Enzymol 216 495 508 3 Schweinfest C W Henderson K W Gu J R Kottaridis S D Besbeas S et al 1990 Genet Anal Tech Appl 7 3 64 70 MSDS INFORMATION Material Safety Data Sheets MSDSs are provided online at Attp www genomics agilent com MSDS documents are not included with product shipments ABLE C Strain and ABLE K Strain Excision Protocol 7
10. the host The ABLE C strain and the ABLE K strain help solve these problems associated with toxic clones The ABLE C strain and the ABLE K strain reduce the copy number of common cloning vectors by 4 and 10 fold from XL 1 BLue respectively enhancing the probability that a toxic clone will be propagated Positive clones observed upon initial screening as lambda plaques can be excised or recloned into any convenient vector and can be introduced into the ABLE strains Alternatively plasmid libraries or excised phagemid libraries can be screened directly in these strains The ABLE strains and a standard high copy number host such as XL1 Blue can be screened simultaneously avoiding the need to repeat cloning operations or to investigate new host vector systems The ABLE strains are completely restriction minus and permit blue white color selection The ABLE strains propagate all ColEl derived vectors e g pBluescript II phagemids pUC pET etc at lower copy numbers thereby reducing the level of the cloned gene product PREPARATION OF HOST CELLS The host strains have been sent as bacterial glycerol stocks For the appropriate media and plates please refer to the following table Host strain ABLE C strain Agar plates for bacterial streak LB kanamycin tetracycline Media for bacterial cultures for titering phage final concentration Media for bacterial glycerol stock LB kanamycin tetracyclines LB supplement
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ABLE C Strain ABLE K Strain Excision Protocol Manual