Manual Perfectprep™ BAC 96 Kit
Contents
1. Manual Perfectprep BAC 96 Kit For rapid and simple purification of BAC PAC fosmid and cosmid DNA in a 96 well format www 5Prime com Manual Perfectprep BAC 96 Kit Trademarks Perfectprep is a trademark of 5 PRIME GmbH VWR and VWR Signature are trademarks of Scientific Holdings Corporation BigDye is a trademark of Applied Biosystems Incorporated Eppendorf and epMotion are registered trademarks of Eppendorf AG 2007 5 PRIME all rights reserved Page 2 www 5Prime com Manual Perfectprep BAC 96 Kit Contents Contents 3 Introduction 4 Precautions and warnings 5 Kit Components Perfectprep BAC 96 Kit 6 Storage and stability 8 Quality assurance 8 Bacterial cultures 8 Vacuum protocol 11 Centrifugation protocol 18 Applications 22 Troubleshooting 25 Additional information 28 References 29 Ordering information 30 5 PRIME Distributors 31 Perfectprep BAC 96 Kit quick centrifugation protocol 33 Perfectprep BAC 96 Kit quick vacuum protocol 35 www 5Prime com Page 3 Manual Perfectprep BAC 96 Kit Introduction Bacterial Artificial Chromosomes BACs have become the vector of choice in the construction of libraries for genome sequencing projects due to their higher stability when compared to other large insert DNA vectors BACs are based on the fertility F factor of Escherichia coli which maintains strict copy number control limiting the number of BACs to 1 2 copies per cell Limiting the2. and 25 ug ml of kanamycin for PACs and cosmids Page 8 www 5Prime com Manual Perfectprep BAC 96 Kit Several other media have been tested using the optimal growth conditions for 2x YT but all resulted in slightly lower DNA yields If LB Luria Bertani broth or 2x LB broth is used the growth time may have to be adjusted to achieve the same DNA yields as 2x YT If richer media such as Superbroth and Terrific Broth are used the bacterial cells will grow to much higher cell densities compared to other media However this can lead to inefficient cell lysis and increased clogging when filtering the bacterial lysate In addition overgrowing these cultures can lead to a higher percentage of dead or starving cells in the culture and the DNA from these preparations may be degraded or contaminated with E coli genomic DNA Therefore Superbroth and Terrific Broth are not recommended for use with the Perfectprep BAC 96 Kit Medium component formulations per liter Culture Medium Components Components Components 2x YT 16 g Tryptone 10 g Yeast extract 5 g NaCl LB 10 g Tryptone 5 g Yeast extract 10 g NaCl 2x LB 20 g Tryptone 10 g Yeast extract 10 g NaCl Inoculation of culture medium The Perfectprep BAC 96 Kit was optimized for BAC DNA purification from cultures seeded directly from glycerol stocks single colonies or from pre cultures of BAC clones Each inoculation method has been tested and produced excellent results in downstream ap
3. www fsg pt VWR International Lda Portugal Rua Alfredo da Silva 3C 1300 040 Lisboa Phone 351 968 753 414 E Mail bioMarke vwr com Web http pt vwr com Contact person Rui Henriques Romania VWR International o z Prievozska 6 821 09 Bratislava Slovakia Phone 4421 2 326 601 31 E Mail roman_kovacik vwr com Web www vwr com Contact person Roman Kovacik www 5Prime com Page 31 Manual Perfectprep BAC 96 Kit 5 PRIME Distributors Serbia amp Montenegro VWR International o z Prievozska 6 821 09 Bratislava Slovakia Phone 421 2 326 601 31 E Mail roman_kovacik vwr com Web www vwr com Contact person Roman Kovacik Sweden VWR International Fagerstagatan 18 A Sp nga SE 163 94 Stockholm Phone 46 708 2138 37 E Mail bioMarke vwr com Web http se vwr com Contact person Lars S derlund Switzerland VWR International AG Lerzenstrasse 16 18 CH 8953 Dietikon Phone 41 44 745 14 77 E Mail bioMarke vwr com Web http ch vwr com Contact person Leila Bouhraoua Slovakia VWR International o z Prievozska 6 821 09 Bratislava Slovakia Phone 421 2 326 601 31 E Mail roman_kovacik vwr com Web www vwr com Contact person Roman Kovacik Slovenia VWR International o z Prievozska 6 821 09 Bratislava Slovakia Phone 421 2 326 601 31 E Mail roman_kovacik vwr com Web www vwr com Contact person Roman Kovacik Spain HUCOA ERL SS C Luis No 9 Edificio Hucoa 28
4. 3001 Leuven Phone 32 16 385 265 E Mail bioMarke vwr com Web http be vwr com Contact person Nico Van Stallen Bosnia Herzegowina VWR International o z Prievozska 6 821 09 Bratislava Slovakia Phone 4421 2 326 601 31 E Mail roman_kovacik vwr com Web www vwr com Contact person Roman Kovacik Bra A distributor will soon be available in your country Please visit our website www 5Prime com Bulgaria VWR International o z Prievozska 6 821 09 Bratislava Slovakia Phone 421 2 326 601 31 E Mail roman_kovacik vwr com Web www vwr com Contact person Roman Kovacik Canada Inter Medico 50 Valleywood Drive Markham Ontario L3R 6E9 Canada Toll free Toll Free English 41 800 387 9643 Toll free Sans Frais Francais 41 800 268 1150 Phone 41 905 470 2520 Fax 41 905 470 2381 E Mail mshackleton inter medico com or info inter medico com Web http www inter medico com Croatia VWR International o z Prievozska 6 821 09 Bratislava Slovakia Phone 421 2 326 601 31 E Mail roman_kovacik vwr com Web www vwr com Contact person Roman Kovacik Czech Republic MEDESA s r 0 Na Vysehrade 1092 57201 POLICKA Phone 420 461 723 555 559 Fax 420 461 723 560 E mail medesa medesa cz Web www medesa cz VWR International o z Prievozska 6 821 09 Bratislava Slovakia Phone 421 2 326 601 31 E Mail roman_kovacik vwr com Web www vwr com Contact person Roman Kovacik Den
5. Perfectprep BAC Solution Mix thoroughly by pipetting up and down or gently reversing the tube before you proceed adding the RNase Solution to the Perfectprep BAC Solution 1 Briefly centrifuge the RNase Solution to collect all liguid in the bottom of the tube Pipet the entire content of the RNase Solution to the Perfectprep BAC Solution 1 including Lysozyme Mix thoroughly by pipetting up and down or gently reversing the tube Page 18 www 5Prime com Manual Perfectprep BAC 96 Kit Prepare appropriate amount of diluted Trapping Buffer One plate Combine 6 ml of the Trapping Buffer Concentrate and 18 ml of 95 100 Isopropanol Mix by inversion and store tightly sealed at room temperature Two plates Combine 12 ml of Trapping Buffer Concentrate and 36 ml 95 100 Isopropanol in a new bottle Mix by inversion and store tightly sealed at room temperature Prepare appropriate amount of diluted Wash Buffer One plate Combine 22 5 ml of Wash Buffer Concentrate and 52 5 ml of 95 100 Ethanol in a new bottle Mix by inversion and store tightly sealed at room temperature Two plates Combine 45 ml of Wash Buffer Concentrate and 105 ml 95 100 Ethanol in a new bottle Mix by inversion and store tightly sealed at room temperature Detailed centrifugation protocol 1 Pellet bacteria by centrifuging the Culture Plate at 1 900 x g for 10 minutes 2 Aftercentrifugation pour off supernatants and blot any remaining medium from the plat
6. Plate Cover plate securely using a Plate Seal Place Culture Plate lid upside down over Plate Seal Apply pressure to the lid with both hands and mix by inversion 5 times Incubate samples for 5 minutes at room temperature Add 200 pl Solution 3 to each well Cover plate securely using a Plate Seal Mix by inversion 10 times as before Place Filter Plate A over a Catch Plate not provided Carefully remove Plate Seal from Culture Plate containing lysate Transfer contents to the corresponding wells of Filter Plate A Centrifuge the Filter Plate A Catch Plate assembly at 1900 x g for 2 minutes Discard Filter Plate A Add 200 ul of diluted Trapping Buffer to each well of the Catch Plate Cover Catch Plate securely with a Plate Seal Place Culture Plate lid upside down over Plate Seal Apply pressure to the lid with both hands and mix by inversion 4 times Incubate for 5 minutes at room temperature www 5Prime com Page 33 Manual Perfectprep BAC 96 Kit 11 12 13 14 15 Place Filter Plate BAC over the Waste Plate used Culture Plate Transfer the Catch Plate contents to the corresponding wells of Filter Plate BAC Centrifuge the Filter Plate BAC Waste Plate assembly at 1 900 x g for 2 minutes Add 600 ul diluted Wash Buffer to each well of Filter Plate BAC Centrifuge the Filter Plate BAC Waste Plate assembly at 1 900 x g for 2 minutes Discard Waste Plate Blot the bottom of the Filter Plate BAC several times on a clea
7. and should not be mistaken for clogging No more than 5 minutes should be spent in trying to get clogged wells to clear through the Filter Plate A Remove the Filter Plate A from the vacuum manifold and discard Open the vacuum manifold lid and carefully remove the Filter Plate BAC and Collection Plate from the vacuum manifold chamber Caution Avoid contacting the bottom of the Filter Plate BAC with any surface during the plate transfers www 5Prime com Page 13 Manual Perfectprep BAC 96 Kit 16 Place the used Culture Plate from step 12 into the vacuum manifold chamber close the vacuum manifold lid and then position the Filter Plate BAC on the manifold lid 17 Add 200 ul of diluted Trapping Buffer to each well of the cleared lysate in the Filter Plate BAC 18 Cover the Filter Plate BAC securely using a Plate Seal Place the Culture Plate lid upside down over the Plate Seal Apply pressure to the lid with both hands and mix the Filter Plate BAC by inversion 4 times Avoid touching drip directors Caution To prevent leakage and cross well contamination it is extremely important to keep constant pressure against the Plate Seal across the entire plate while mixing 19 Place the Filter Plate BAC on the manifold lid and incubate for 5 minutes at room temperature 20 Carefully remove the Plate Seal from the Filter Plate BAC then slowly apply vacuum as described in step 13 until all liguid flows through the Filter Plate B
8. for 5 minutes at room temperature 9 Add 200 ul of Solution 3 to each well of the Culture Plate to neutralize the bacterial lysate Cover the Culture Plate securely using a Plate Seal 10 Mix by inversion 10 times as in Step 7 11 Place the Filter Plate A over a 96 well Catch Plate capable of holding at least 2 ml not provided 12 Carefully remove the Plate Seal from the Culture Plate and save the Culture Plate to be used later as a Waste Plate Transfer contents neutralized lysate from the Culture Plate to the corresponding wells of the Filter Plate A Transfer as much of the contents of the Culture Plate as possible by setting the pipette to 750 ul Cover the Filter Plate A securely using a Plate Seal Pipetting lysate to the side of the wells is recommended Transfer of white cell debris to the Filter Plate A is not a problem and will occur Save the used Culture Plate for a Waste Plate in step 17 13 Centrifuge the Filter Plate A Catch Plate assembly at 1 900 x g for 2 minutes Check to make sure all of the neutralized lysate has passed through the Filter Plate A If lysate has not completely passed through the Filter Plate A centrifuge at 1 900 x g for an additional 2 minutes Discard the Filter Plate A after centrifugation 14 Add 200 ul of diluted Trapping Buffer to each well of the Catch Plate containing the cleared lysate 15 Cover the Catch Plate securely using a Plate Seal Place a Culture Plate lid upside down over
9. lid Be certain the plates are in the same orientation Carefully peel off and discard the Plate Seal from the Culture Plate Transfer contents neutralized lysate from the Culture Plate to the corresponding wells of the Filter Plate A Set the pipette to 750 ul and transfer as much of the contents from the original Culture Plate as possible Note Pipetting lysate to the sides of the wells is recommended Transfer of white cell debris to the Filter Plate A will occur and is not a problem Save the used Culture Plate for a Waste Plate in Step 16 It may be necessary to move the pipet tip back and forth to collect as much lysate as possible When using the Single Vac or the Ouad Vac manifold turn on the vacuum pump and apply vacuum by switching vacuum valve to the open position and turning the bleed valve clockwise It may be necessary to apply light pressure to the top of the plate by pressing down to engage the vacuum Vacuum until all liguid neutralized lysate flows through the Filter Plate A Slowly bleed the vacuum by turning bleed valve counterclockwise Allow the vacuum manifold chamber pressure to egualize to ambient pressure Vacuum valve can now be switched to the closed position Note If some wells of the Filter Plate A clog cover plate with a Plate Seal while the vacuum is being drawn Covering the plate with a Plate Seal will increase the vacuum pressure Some appearance of white flocculent material in wells is normal
10. the dark e Centrifuge the plate at 3 000 x g for 30 minutes at 4 C or 1 900 x g for 45 minutes at 4 C f Pour off the supernatant into the sink with a flicking motion of the plate and then blot the plate on clean absorbent material g Invert the plate unsealed onto 2 3 layers of clean absorbent material and centrifuge at 50 x g for 1 minute to remove any residual ethanol h Add 150 ul of cold 20 C 70 high purity ethanol to each well of the plate using a multi channel pipet and seal using a plate seal i Centrifuge the plate at 1 900 x g for 15 minutes at 4 C j Pour off the supernatant into the sink k Invert the plate unsealed onto 2 3 layers of clean absorbent material and centrifuge at 50 x g for 1 minute to remove any residual ethanol Air dry the plate for 10 minutes in the dark Seal the plate using a plate seal and store at 20 C until ready to sequence www 5Prime com Page 23 Manual Perfectprep BAC 96 Kit 7 Resuspend the pellets in 8 ul of 0 1x TE immediately prior to sequencing 8 Run the samples on an ABI 3700 DNA Sequencer using a 90 second injection time Sequencing data The following data was generated using BAC clones from plate 707 of the CITB Human BAC library Invitrogen Research Genetics Inc DNA from the 384 different clones was purified using the Perfectprep BAC 96 Kit according to the vacuum protocol and standard elution method described in this user manual 10 ul of BAC DNA temp
11. to re dissolve Prepare the complete Perfectprep BAC Solution 1 Perfectprep BAC Solution 1 plus RNase A Solution and lyophilized Lysozyme 1 Resuspend the lyophilized Lysozyme in the following amount of Perfectprep BAC Solution Kit Name Perfectprep BAC Perfectprep BAC Perfectprep BAC 96 96 Kit 2 Plts 96 Kit 10 Plts Base Kit 50 Lysozyme 40 mg 250 mg 2 x 625 mg lyophilized Add Perfectprep 1 8 ml 7ml 2x14 ml BAC Solution 1 2 Mix thoroughly by pipetting up and down or gently reversing the tube Take care to ensure that all of the powder is dissolved Some foaming will occur 3 Pipet the entire content of the Lysozyme Solution to the Perfectprep BAC Solution Mix thoroughly by pipetting up and down or gently reversing the tube before you proceed adding the RNase Solution to the Perfectprep BAC Solution 1 4 Briefly centrifuge the RNase Solution to collect all liguid in the bottom of the tube 5 Pipet the entire content of the RNase Solution to the Perfectprep BAC Solution 1 including Lysozyme Mix thoroughly by pipetting up and down or gently reversing the tube Prepare appropriate amount of diluted Trapping Buffer One plate Combine 6 ml of the Trapping Buffer Concentrate and 18 ml of 95 100 Isopropanol Mix by inversion and store tightly sealed at room temperature Two plates Combine 12 ml of Trapping Buffer Concentrate and 36 ml 95 100 Isopropanol in a new bottle Mix by inversion and store tightl
12. 031 Madrid Phone 34 91 380 67 10 Fax 34 91 380 85 02 E Mail atc amp hucoa erloss com Web www hucoa erloss com WR International Eurolab S L Apartado 48 E 08100 Mollet del Valles Phone 434 660 398 304 E Mail bioMarke vwr com Web http es vwr com Contact person Monica Galiana Thailand Mondotech Thailand Co Ltd 88 Krungthepkreetha Rd Huamark Bangkapi Bangkok 10240 Phone 66 2 379 4212 5 Fax 66 2 379 4216 E Mail mondotec asianet co th Contact person Mr Tosphon Pruchyathamkorn or Ms Yalada Chullakasevee USA Fisher Scientific Company LLC Phone 41 800 766 7000 Fax 41 800 926 1166 Web www fishersci com For all other countries Visit our website www 5Prime com or contact us 5 PRIME GmbH K nigstr 4a 22767 Hamburg Germany Phone 49 40 3197 927427 5 PRIME Inc 111 Bucksfield Road Gaithersburg MD 20878 US Phone 1 240 683 3905 Page 32 www 5Prime com Manual Perfectprep BAC 96 Kit Perfectprep BAC 96 Kit quick centrifugation protocol Inoculate 1 5 ml 2x YT medium Grow culture according to protocol Centrifuge at 1 900 x g for 10 minutes to pellet cells Pour off supernatant after centrifugation Blot plate on absorbent material to remove residual culture medium Add 200 ul Solution 1 to each well of Culture Plate Cover plate securely using a Plate Seal Resuspend pellets by vigorously vortexing the plate 2 3 minutes Add 200 ul Solution 2 to each well of Culture
13. 2 326 601 31 E Mail roman_kovacik vwr com Web www vwr com Contact person Roman Kovacik Lithuania VWR International o z Prievozska 6 821 09 Bratislava Slovakia Phone 4421 2 326 601 31 E Mail roman_kovacik vwr com Web www vwr com Contact person Roman Kovacik Luxembourg VWR International BVBA SPRL Haasrode Researchpark Zone 3 Geldenaaksebaan 464 B 3001 Leuven Phone 32 16 385 265 E Mail bioMarke vwr com Web http be vwr com Contact person Nico Van Stallen New Zealand Global Science and Technology 241 Bush Road Albany 0632 Auckland Phone 64 9 443 5867 Fax 64 9 444 7314 E Mail global globalscience co nz Web www globalscience co nz Netherlands VWR International B V Postbus 8198 NL 1005 AD Amsterdam Phone 31 20 4800 8640 E Mail bioMarke vwr com Web http nl vwr com Contact person Esther Koornneef Norway VWR International AS Kakkelovnskroken 1 P B 45 Kalbakken NO 0901 Oslo Phone 47 908 236 78 E Mail bioMarke vwr com Web http no vwr com Contact person Nina Nielsen Bjerke Poland VWR International o z Prievozska 6 821 09 Bratislava Slovakia Phone 4421 2 326 601 31 E Mail roman_kovacik vwr com Web www vwr com Contact person Roman Kovacik Portugal FSG Grupo Hucoa Erl ss Edificio EURO Rua Pedro Alvares Cabral 24 3 D 2670 391 Infantado Phone 351 21 425 3350 Fax 351 21 425 3351 E Mail achambel hucoa erloss com Web
14. 20 Collection Plates 50 Pit 2300230 Culture Plates 50 Plt 2300240 Perfectprep BAC Solution 1 850 ml 2300330 Perfectprep BAC Solution 2 850 ml 2300340 Perfectprep BAC Solution 3 850 ml 2300350 Perfectprep Plasmid 96 Vac Kit 2 Plt 2300200 Perfectprep Plasmid 96 Vac Kit 10 Plt 2300210 Perfectprep Plasmid 96 Vac Base Kit 50 Pl 2300220 Perfectprep Vac Solution 1 1000 ml 2300250 Perfectprep Vac Solution 2 1000 ml 2300260 Perfectprep Vac Solution 3 1000 ml 2300270 Perfectprep Vac Manifold single basic 2300280 Perfectprep Vac Manifold single 2300290 1without plates please order Culture Plate Catalog No 2300240 and Collection Plate Catalog No 2300230 separately Page 30 www 5Prime com Manual Perfectprep BAC 96 Kit 5 PRIME Distributors Australia Quantum Scientific 30 Finchley Street Milton Qld 4064 P O Box 164 Paddington Queensland 4064 Phone 61 7 3369 9544 Fax 61 7 3368 1802 E Mail salesequantum scientific com au Web www quantum scientific com au Austria Schott Lab Technology GmbH Br nner StraBe 73 1210 Wien Phone 43 1 29017560 Fax 43 1 290175620 E Mail info austria schott com Web http www schott labtech com VWR International GmbH Graumanngasse 7 1150 Wien Phone 43 1 97 002 328 E Mail bioMarke vwr com Web http at vwr com Contact person Brigitte Niebler Belgium VWR International BVBA SPRL Haasrode Researchpark Zone 3 Geldenaaksebaan 464 B
15. AC Bleed the vacuum as described in step 14 and allow the vacuum manifold chamber pressure to egualize to ambient pressure The BAC DNA molecules are now trapped on the surface of the membrane in the Filter Plate BAC 21 Add 600 ul of diluted Wash Buffer to each well of the Filter Plate BAC Slowly apply vacuum until all of the liguid has passed through the Filter Plate BAC Bleed the vacuum and allow the vacuum manifold chamber pressure to egualize to ambient pressure 22 Remove the Filter Plate BAC from the vacuum manifold and thoroughly blot the drip directors of the Filter Plate BAC several times on a clean absorbent material Wipe excess liguid from around the edges of the bottom of the Filter Plate BAC with an absorbent material Note Failure to blot the bottom of the Filter Plate BAC to remove excess liguid may result in unwanted reagent contamination of the BAC eluate This could negatively affect the performance of the BAC DNA in downstream applications 23 Open the vacuum manifold lid and remove the used Culture Plate from the vacuum manifold chamber and discard 24 Place the Filter Plate BAC over the empty manifold chamber and allow it to air dry for 5 minutes 25 Blot the drip directors of the Filter Plate BAC several times on a clean absorbent material to eliminate any ethanol remaining from the diluted Wash Buffer Page 14 www 5Prime com Manual Perfectprep BAC 96 Kit 26 Place a Tall Adapter Plate provided wit
16. Filter Plate BAC Note Liguid may appear to pass through the Filter Plate BAC membrane guickly it is critical that vacuum pressure reach at least 15 inches of mercury before bleeding Monitoring vacuum pressure will ensure that all liguid has passed through the membrane and into the Collection Plate 4 Bleed vacuum and allow vacuum chamber pressure to egualize to ambient pressure 5 Apply a second 60 ul aliquot of Elution Buffer to the center of each well just above the membrane of the Filter Plate BAC Apply vacuum as in step 3 until the Elution Buffer has passed through the Filter Plate BAC then slowly bleed the vacuum and allow the vacuum manifold chamber pressure to egualize to ambient pressure 6 Apply a third 60 pl aliquot of the Elution Buffer to the center of each well just above the membrane of the Filter Plate BAC Apply vacuum as in step 3 until the Elution Buffer has passed through the Filter Plate BAC Slowly bleed the vacuum and allow the vacuum manifold chamber pressure to egualize to ambient pressure 7 Dry BAC DNA samples in the Collection Plate for approximately 3 3 5 hours at 60 C in a SpeedVac with a plate rotor or in a vacuum chamber If a SpeedVac or vacuum chamber is not accessible samples may be dried down overnight in an incubator set at 37 C Note Prior to drying in a regular vacuum chamber or incubator the plate must be centrifuged briefly at 1 000 x g Liguid should no longer be visible in the wells whe
17. Longer initial denaturing times are reguired for BACs due to the large length of the genomic DNA inserts We have found that denaturing the template DNA for 5 minutes at 95 C prior to adding the seguencing reaction master mix as described in Section Seguencing on page 22 of this user manual yields longer seguencing read lengths Read lengths may vary for a variety of large insert DNA clones depending on the host strain type of vector and the length of the genomic DNA insert In addition several factors such as the amount of template the amount of primer the reaction volume size the length of the cycling extension step and the number of cycles can all affect the sequencing read length results Optimization experiments may be necessary to determine the ideal conditions for the clones being sequenced Page 27 Manual Perfectprep BAC 96 Kit Additional information 5 PRIME vacuum manifolds 5 PRIME offers two single position vacuum manifold systems Perfectprep Vac Manifold Single Basic and Perfectprep Vac Manifold Single The manifolds can be used with a vacuum pump or a house vacuum if sufficient vacuum can be achieved Normal atmospheric pressure is approximately 101 kPa or 30 inches of mercury The vacuum source must be able to create a vacuum of approximately 70 kPa or 20 inches of mercury Please see page 30 for ordering information Pressure conversions To convert from kiloPascals kPa to Multiply by Millim
18. ce and then washed resuspended and eluted into a collection plate The eluted BAC DNA is immediately ready for use in downstream applications The Perfectprep BAC 96 Kit provides enough template for at least four BAC end sequencing reactions and one fingerprint analysis The purified BAC DNA is suitable for use in the following downstream applications Sequencing Fingerprinting Mutagenesis PCR Page 4 www 5Prime com Manual Perfectprep BAC 96 Kit Precautions and warnings Appropriate safety apparel such as lab coat gloves and eye protection should be worn The following risk and safety phrases apply to components of the Perfectprep BAC 96 Kit Solution 2 Corrosive contains Sodium Hydroxide R34 526 536 37 39 545 Solution 3 Irritant R36 38 S36 37 39 For more information please consult the appropriate material safety data sheets which are available for this kit online at www 5Prime com msds Risk and safety phrases R34 Causes burns R36 38 Irritating to eyes and skin 26 In case of contact with eyes rinse immediately with plenty of water and seek medical advice S36 37 39 Wear suitable protective clothing gloves and eye face protection S45 In case of accident or if you feel unwell seek medical advice immediately show the label where possible www 5Prime com Page 5 Manual Perfectprep BAC 96 Kit Kit Components Perfectprep BAC 96 Kit Contents Perfectprep BAC Perfectprep BAC Perfectpr
19. copy number minimizes the risk of DNA recombination while maintaining a stable cloned DNA insert within the host cell However due to the low number of BACs per cell obtaining sufficient guantities of high guality BAC DNA when using lower reagent and starting material volumes has posed a challenge for researchers 5 PRIME has solved this problem with the development of the Perfectprep BAC 96 Kit which allows parallel processing of 96 samples The Perfectprep BAC 96 Kit is specifically designed for the purification of BAC DNA from E coli bacterial cultures grown and processed in a 96 well plate format Processing may be performed by using either a full vacuum or a centrifugation protocol One 96 well plate can be processed manually in approximately 60 minutes and two plates in 60 75 minutes The Perfectprep BAC 96 Kit is also compatible with isolation of other large circular constructs such as PACs fosmids and cosmids The versatility of the Perfectprep BAC 96 Kit enables easy integration onto automated liquid handling workstations such as the epMotion 5075 Vac from Eppendorf or equivalent The Perfectprep 96 BAC chemistry is also available in a high throughput format 50 plate kit for processing a large number of plates The Perfectprep 96 BAC protocol uses a modified alkaline lysis procedure in which bacterial cells are lysed in the presence of RNase A Using a proprietary technology the liberated BAC DNA is trapped on a membrane surfa
20. e by inverting and tapping the plate on a clean absorbent material Residual medium left in the wells can negatively affect the prep 3 Pelleted bacterial cells can be used immediately covered with a Plate Seal and stored at 4 C for up to four hours or 20 C for up to two months It is recommended to use bacterial cells immediately 4 Add 200 ul of Solution 1 to the cell pellets in each well of the Culture Plate Cover securely using a Plate Seal 5 Resuspend cell pellets by vortexing the plate for 2 3 minutes on medium high setting to resuspend the cells After vortexing for 2 minutes check for complete dispersion of the pellet If the pellets are not clearly resuspended the plate can be vortexed an additional minute Note Complete resuspension of the pellets is critical to obtaining high yields of pure BAC DNA Insufficient resuspension can result in processing problems such as clogging of the Filter Plate A in step 13 6 Add 200 pl of Solution 2 to each well of the Culture Plate to lyse cells Cover securely using a Plate Seal www 5Prime com Page 19 Manual Perfectprep BAC 96 Kit 7 Place the Culture Plate lid upside down over the Plate Seal Apply pressure to the lid with both hands and mix the Culture Plate by gently inverting 5 times Caution While mixing it is extremely important to keep constant pressure against the Plate Seal over the entire plate to prevent leakage and cross well contamination 8 Incubate
21. elution method 4 Add the appropriate volume of master mix to each sample so that the total reaction volume is 20 pl Seal plate mix well and centrifuge briefly Page 22 www 5Prime com Manual Perfectprep BAC 96 Kit 5 Cycle the reactions according to the following program a 95 C for 5 minutes b 95 C for 15 seconds c 50 55 C for 15 seconds d 60 C for 4 minutes e Repeat steps b d for 100 cycles f Hold at 4 C 10 C 6 Clean up sequencing reactions using the following protocol optimized for sequencing on the ABI 3700 Before starting A refrigerated centrifuge is required for this protocol Verify that the temperature of the centrifuge is set at 4 C Turn on the heat sealer for sealing plate before vortexing Prepare 70 high purity ethanol and chill to 20 C Note BigDye Terminator chemistry is light sensitive A dark area such as a drawer should be available for the incubation steps a Add 5 ul of 125 mM EDTA to each well of the plate using a multi channel pipet Note The EDTA must be directly added to the the reaction mix prior to proceeding to the next step If necessary gently tap the plate to ensure that the EDTA combines with the reaction mix b Add 60 ul of 95 100 high purity ethanol to each well of the plate using a multichannel pipet c Seal the plate using a heat sealer and thoroughly vortex the plate for 8 10 seconds d Incubate the plate at room temperature for 25 minutes in
22. ep BAC 96 Kit 2 Plt 96 Kit 10 Plt 96 Base Kit 50 Catalog number 2300300 2300310 2300320 Number of Samples 192 960 4800 Processed Rini 45 ml 300 ml 2 x 850 ml Solution 1 Perfectprep BAC 45 ml 300 ml 2 x 850 ml Solution 2 EE IR CR EAC 45 ml 300 ml 2 x 850 ml Solution 3 0 5 ml 0 3 ml 2 x 0 85 ml 2 RNase A Solution 10 mg ml 100 mg ml 100 mg ml Lysozyme lyophilized 40 mg 250 mg 2 x 625 mg Perfectprep BAC Trapping Buffer 12 ml 75 ml 2 x250 ml Concentrate Pane ii Wed aS 2 x 150 ml 3 x 600 ml Buffer Concentrate Perfectprep BAC Elution Buffer 60 ml 260 ml 750 ml Culture Plate 2 5x2 Filter Plate A 2 5x2 25x2 Perfectprep BAC Filter Plate BAC Z 2x2 dia Collection Plate 2 5x2 Plate Seals 25 3x25 3x25 Perfectprep BAC 2 5x2 Air Permeable Seals 1without plates please order Culture Plate Catalog No 2300240 and Collection Plate Catalog No 2300230 separately 2 please see pages 11 and 18 before starting the protocol Page 6 www 5Prime com Manual Perfectprep BAC 96 Kit Additional Materials Required For vacuum and centrifugation protocols Plate shaker or vortexer for cell resuspension Note Any plate shaker or vortexer can be used however the Perfectprep 96 Bac protocols were optimized using the VWR Signature Multi tube Vortexer from VWR at setting 7 approximately 1 900 cycles per minute Liquid bacterial growth medium and the appropriate antibiotic see page 8f 95 100 ethanol f
23. erfectprep BAC 96 Kit provides a fast and robust method for purifying BAC DNA to be used in applications such as DNA sequencing The sequencing protocol below has been optimized for direct sequencing of BAC DNA on an ABI 3700 DNA sequencer It incorporates the latest ABI BigDye advances and includes an improved reaction cleanup method providing even better sequencing performance A standard 60 ul elution from the Perfectprep 96 Bac procedure provides enough BAC DNA for at least five seguencing reactions Seguencing has been optimized using 1 4 BigDye Terminator v3 1 reactions in a 20 ul reaction volume Excellent results have been observed using 10 ul of template from the standard elution method independent of DNA concentration Please see the Seguencing Data Section for examples of performance 1 Add 5 10 ul of BAC DNA to a thermal cycling plate 2 Denature the samples in a thermal cycler for 5 minutes at 95 C then place immediately on ice until the addition of the seguencing reaction master mix 3 Prepare the seguencing reaction master mix as follows Reagent Volume per reaction BAC DNA Template 5 10 pl 5x Sequencing Buffer ABI 3 pl BigDye Terminator Ready Reaction Premix v3 1 ABI 2 pl Primer 10 uM 1 ul MgCl 25 mM 0 6 ul H20 QS to 20 pl Total Volume 20 pl 10 pl of template is used when kit is processed using the standard elution method 5 pl of template may be used when kit is processed using the optional
24. eters of Mercury mm Hg 7 500638 Inches of Mercury in Hg 0 2953 Atmospheres atm 0 009869 Torrs Torr 7 500638 Millibars mbars 10 Pounds per sguare inch psi 0 145038 Page 28 www 5Prime com Manual Perfectprep BAC 96 Kit References 1 Birnboim H C and Doly J 1979 A rapid alkaline lysis procedure for screening recombinant plasmid DNA Nucleic Acids Res 7 1513 1522 2 Ewing B and Green P 1998 Base calling of automated sequencer traces using phred II Error probabilities Genome Res 8 186 194 3 Ewing B Hillier L Wendl M C and Green P 1998 Base calling of automated sequencer traces using phred I Accuracy Assessment Genome Res 8 175 185 4 Marra M A Kucaba T A Dietrich N L Green E D Brownstein B Wilson R K McDonald K M Hillier L W McPherson J D and Waterson R H 1997 High throughput fingerprint analysis of large insert clones Genome Res 7 1072 1084 5 Shizuya H Birren B Kim U Mancino V Slepak T Tachiiri Y and Simon M 1992 Cloning and stable maintenance of 300 kilobase pair fragments of human DNA in Escherichia coli using an F factor based vector Proc Natl Acad Sci 89 8794 8797 www 5Prime com Page 29 Manual Perfectprep BAC 96 Kit Ordering information Product Package Size Catalog No Perfectprep BAC 96 Kit 2 Plt 2300300 Perfectprep BAC 96 Kit 10 Plt 2300310 Perfectprep BAC 96 Base Kit 50 Plt 23003
25. h manifold inside the manifold chamber Place an unused Collection Plate on top of a Tall Adapter Plate and close the manifold lid Place the Filter Plate BAC containing the trapped BAC DNA on the manifold lid Verify that both plates are in the same orientation Important Procedure Note Steps 27 34 outline the standard elution method for this protocol An optional elution method for increased yield and concentration is also available and is outlined on page 16 All downstream application procedures provided in this manual were performed using the standard elution method 27 Add 30 pl of Elution Buffer to each well of the Filter Plate BAC Add the Elution Buffer to the center of each well just above the membrane Note To avoid inconsistent elution volumes ensure that the Elution Buffer is pipetted onto the surface of the filter and not the side of the well 28 Incubate for 5 minutes 29 Apply vacuum by turning bleed valve clockwise until completely closed Vacuum until all liguid passes through the Filter Plate BAC Note Liguid may appear to pass through the Filter Plate BAC membrane guickly it is critical that vacuum pressure reach at least 15 inches of mercury before bleeding Monitoring vacuum pressure will ensure that all liguid has passed through the Filter Plate BAC and into the Collection Plate 30 Bleed vacuum and allow vacuum chamber pressure to egualize to ambient pressure 31 Apply a second 30 ul aliguot of Elutio
26. he Eppendorf centrifuges 5804 and 5810 and A 2 DWP Rotor or equivalent for use with centrifugation protocol Storage and stability Store all Perfectprep BAC 96 Kit components tightly sealed at room temperature Do not freeze All non diluted Perfectprep BAC 96 Kit components are stable for at least 12 months when stored unopened as described above Quality assurance Each lot of the Perfectprep BAC 96 Kit is functionally tested by isolating two different human BAC clones from E coli cultures as described in the vacuum protocol The purified BAC DNA is tested for quantity and quality by agarose gel electrophoresis and for performance in automated fluorescent cycle sequencing Bacterial cultures Culture media 2x YT broth is the recommended culture medium for cultivating BAC clones with the Perfectprep BAC 96 Kit This medium produces high cell densities and leads to excellent yields of low copy number BAC DNA Optimal growth conditions 24 hours at 37 C and 325 rpm were determined using human BAC clones from the RPCI 11 library Factors such as host strain and type of vector can drastically affect the yield of BAC DNA Thus growth conditions may need to be adjusted depending on the clones being purified The appropriate antibiotic should be added to the medium depending upon the antibiotic resistance gene present in the vector of the clones being grown The recommended concentrations are 12 5 ug ml of chloramphenicol for BACs and fosmids
27. he bottom of the Filter Plate BAC to remove excess liguid may result in unwanted reagent contamination of the BAC eluate This could negatively affect the performance of the BAC DNA in downstream applications Place the Filter Plate BAC over an unused Collection Plate Add 30 pl of Elution Buffer to each well of the Filter Plate BAC Add the Elution Buffer to the center of each well just above the membrane Note To avoid inconsistent elution volumes ensure that the Elution Buffer is pipetted onto the surface of the filter and not the sides of the wells Incubate for 5 minutes at room temperature Centrifuge the Filter Plate BAC Collection Plate assembly at 1 900 x g for 2 minutes Apply a second 30 ul aliquot of Elution Buffer to the center of each well just above the membrane of the Filter Plate BAC Centrifuge the Filter Plate BAC Collection Plate assembly at 1 900 x g for 2 minutes Remove the Filter Plate BAC Collection Plate assembly from the centrifuge and discard the Filter Plate BAC DNA is immediately ready for use in downstream applications or may be stored To store purified BAC DNA cover Collection Plate containing the purified BAC DNA using a Plate Seal and store at 20 C www 5Prime com Page 21 Manual Perfectprep BAC 96 Kit Applications Sequencing BAC vectors are widely used in genome sequencing projects and provide the foundation for construction of BAC libraries used in genome mapping and sequencing The P
28. ifficult to sequence Excess BigDye terminator loaded onto sequencer Page 26 Proper cultivation of BAC clones is essential for obtaining sufficient quantities of high quality template DNA Follow the inoculation and growth conditions outlined in Sections 7 1 7 3 of this user manual Redesign sequencing primers Make sure sequencing reactions are properly cleaned up to purify the extension products before loading the samples on a DNA sequencer www 5Prime com Manual Perfectprep BAC 96 Kit Poor DNA sequencing cont Comments Suggestions Ethanol contamination in the eluted DNA from not drying the Filter Plate BAC properly BAC DNA not sufficiently denatured before cycle sequencing Sub optimal sequencing reaction mix conditions and cycle sequencing parameters www 5Prime com If the bottom of the Filter Plate BAC is not thoroughly dried according to Steps 21 24 in the Vacuum protocol residual ethanol from the diluted Wash Buffer on the Filter Plate BAC may be carried over in the subseguent elution steps If this occurs the ethanol in the eluted DNA will inhibit cycle seguencing reactions resulting in shorter seguencing read lengths Make sure the drip directors of the Filter Plate BAC are thoroughly blotted on clean absorbent material and excess diluted Wash Buffer is wiped from the edges of the bottom of the Filter Plate BAC Ensure that the Filter Plate BAC is air dried for 5 minutes
29. l Mix by inversion 10 times as before 8 Prepare manifold for the first vacuum step Place a short adaptor in the manifold chamber Position the Filter Plate BAC over the short adaptor Close the lid of the manifold Position the Filter Plate A on the manifold lid Be certain the plates are in the same orientation 9 Remove Plate Seal from the Culture Plate 10 Transfer Culture Plate contents to the corresponding Filter Plate A wells and set Culture Plate aside to be used later as Waste Plate www 5Prime com Page 35 Manual Perfectprep BAC 96 Kit 11 Apply vacuum by switching vacuum valve to the open position and turning bleed valve clockwise Vacuum until all liquid has passed through Filter Plate A Slowly bleed vacuum by turning bleed valve counter clockwise Allow the vacuum manifold chamber pressure to equalize to ambient pressure Turn vacuum valve to the off position 12 Remove Filter Plate A from manifold and discard Remove Filter Plate BAC and Collection Plate from the manifold Place used Culture Plate into the vacuum manifold chamber and close manifold lid Place Filter Plate BAC on the manifold lid 13 Add 200 ul diluted Trapping Buffer to Filter Plate BAC Cover Filter Plate BAC securely with a Plate Seal Place the Culture Plate lid upside down over the Plate Seal Apply pressure to the lid with both hands and invert 4 times Avoid touching drip directors Place Filter Plate BAC on manifold lid Incubate for 5 minutes at room tem
30. late was cycle seguenced using universal end sequencing primer T7 5 TAA TAC GAC TCA CTA TAG GG 3 Cycling clean up and seguencing were performed based on the protocols and parameters described in Section Seguencing on page 22 Ouality scoring was performed using Phred base calling software CodonCode Corporation Passing scores were determined to be those samples with read lengths of greater than 100 Phred O gt 20 bases Average seguencing data from BAC Clones Average Phred O gt 20 96 Passing samples bases Plate 1 673 93 8 Plate 2 710 99 Plate 3 702 93 8 Plate 4 672 99 Fingerprinting BAC DNA fingerprinting uses restriction enzymes to digest the DNA and generate a number of DNA fragments that can be separated by agarose gel electrophoresis Fingerprinting data from BAC clones are analyzed to determine which clones share a high percentage of the same size fragments and therefore the extent to which the clones overlap From this a contiguous series of overlapping clones is constructed providing a detailed map to determine which BAC library clones are appropriate for sequencing This is an effective method for organizing large genome sequencing project data BAC DNA purified using the Perfectprep BAC 96 Kit can be used immediately in fingerprinting reactions We have found that 10 ul of Perfectprep BAC DNA can be completely digested with as little as 5 units of enzyme in a 20 pl reaction Page 24 www 5Prime com Man
31. mark VWR International ApS Valhojs All 174 176 2610 Redovre Phone 45 21 22 37 60 E Mail bioMarke vwr com Web http dk vwr com Contact person Nikolaj Schmolker Estonia VWR International o z Prievozska 6 821 09 Bratislava Slovakia Phone 421 2 326 601 31 E Mail roman_kovacik vwr com Web www vwr com Contact person Roman Kovacik Finland VWR International Oy Pihat rm 1 C1 FI 02240 Espoo Phone 358 9 804 55 209 E Mail bioMarke vwr com Web http fi vwr com Contact person Leena Kontola Kuusisto France Dominigue Dutscher SA 30 rue de l Industrie P O Box 62 67172 Brumath Cedex Phone 33 3 88 59 33 90 Fax 33 3 88 59 33 99 E Mail info dutscher com Web www dutscher com VWR International S A S Le P rigares B timent B 201 rue Carnot F 94126 Fontenay sous Bois Cedex Phone 33 1 45 14 83 43 E Mail bioMarke vwr com Web http fr vwr com Contact person Soraya Mezaib Germany VWR International GmbH HilpertstraBe 20a D 64295 Darmstadt Phone 49 6151 3972 442 E Mail bioMarke vwr com Web http de vwr com Contact person Matthias Dornheim Greece VWR International o z Prievozska 6 821 09 Bratislava Slovakia Phone 421 2 326 601 31 E Mail roman_kovacik vwr com Web www vwr com Contact person Roman Kovacik Fax 44 1159 455 379 E Mail flowgensales flowgen co uk Web www Flowgen co uk Great Britain FLOWGEN Wilford Industrial Estate Ruddingt
32. n Buffer to the center of each well just above the membrane of the Filter Plate BAC 32 Apply vacuum as in step 29 until the Elution Buffer has passed through the Filter Plate BAC Slowly bleed the vacuum and allow the vacuum manifold chamber pressure to egualize to ambient pressure Remove the Filter Plate BAC from the vacuum manifold and discard 33 Open the vacuum manifold lid and carefully recover the Collection Plate from the vacuum manifold chamber 34 DNA is immediately ready for use in downstream applications or may be stored To store purified BAC DNA cover the Collection Plate containing the purified BAC DNA using a Plate Seal and store at 20 C www 5Prime com Page 15 Manual Perfectprep BAC 96 Kit Optional elution method The following optional elution method can be used when increased yield and concentration are required A dry down step is required following elution to reduce the final volume of the eluate and concentrate the DNA Begin this procedure after step 26 of the vacuum protocol 1 Add 60 ul of Elution Buffer to each well of the Filter Plate BAC Add the Elution Buffer to the center of each well just above the membrane Note To avoid inconsistent elution volumes ensure that the elution buffer is pipetted onto the surface of the filter and not the side of the well 2 Incubate for 5 minutes Apply vacuum by turning bleed valve clockwise until completely closed Vacuum until all liguid passes through the
33. n absorbent material and wipe excess liquid from around the bottom edges of the Filter Plate BAC Place the Filter Plate BAC over a Collection Plate Add 30 ul of Elution Buffer to the center of each Filter Plate BAC well Incubate 5 minutes at room temperature Centrifuge the Filter Plate BAC Collection Plate assembly at 1 900 x g for 2 minutes Apply a second 30 ul aliquot of Elution Buffer to the center of each Filter Plate BAC well Centrifuge the Filter Plate BAC Collection Plate assembly at 1 900 x g for 2 minutes DNA is ready to use in downstream applications Page 34 www 5Prime com Manual Perfectprep BAC 96 Kit Perfectprep BAC 96 Kit quick vacuum protocol 1 Inoculate 1 5 ml 2x YT medium Grow culture according to protocol 2 Centrifuge at 1900 x g for 10 minutes to pellet cells 3 Pour off supernatant after centrifugation 4 Blot plate on absorbent material to remove residual culture medium 5 Add 200 ul Solution 1 to each well of Culture Plate Cover plate securely using a Plate Seal Resuspend pellets by vigorously vortexing the plate 2 3 minutes 6 Add 200 ul Solution 2 to each well of Culture Plate Cover plate securely using a Plate Seal Place Culture Plate lid upside down over Plate Seal Apply pressure to the lid with both hands and mix by inversion 5 times Incubate samples for 5 minutes at room temperature 7 Add 200 ul Solution 3 to each well to neutralize lysate Cover plate securely using a Plate Sea
34. n medium high setting to resuspend the cell pellets After vortexing for 2 minutes check for complete dispersion of the pellet If the cells are not clearly resuspended the plate can be vortexed an additional minute Note Complete resuspension of the pellets is critical to obtaining high yields of pure BAC DNA Insufficient resuspension can result in downstream processing problems that could cause clogging of Filter Plate A in Step 14 6 Add 200 ul of Solution 2 to each well of the Culture Plate to lyse cells Cover plate securely using a Plate Seal 7 Place the Culture Plate lid upside down over the Plate Seal Apply pressure to the lid with both hands and mix the Culture Plate by inversion 5 times Caution To prevent leakage and cross well contamination it is extremely important to keep constant pressure against the Plate Seal across the entire plate while mixing Page 12 www 5Prime com Manual Perfectprep BAC 96 Kit 10 11 12 13 15 Incubate for 5 minutes at room temperature Add 200 ul of Solution 3 to each well of the Culture Plate to neutralize the bacterial lysate Cover the Culture Plate securely using a Plate Seal Mix by inversion 10 times as in step 7 Prepare the manifold for the first vacuum step Place a short adaptor provided with the manifold in the manifold chamber Position the Filter Plate BAC over the short adaptor Close the lid of the manifold Position the Filter Plate A on the manifold
35. n the samples are completely dry Page 16 www 5Prime com Manual Perfectprep BAC 96 Kit 8 Resuspend the samples in 30 ul of Molecular Biology Grade Water 9 DNA is immediately ready for use in downstream applications or may be stored To store purified BAC DNA Cover the Collection Plate containing the purified BAC DNA using a Plate Seal and store at 20 C www 5Prime com Page 17 Manual Perfectprep BAC 96 Kit Centrifugation protocol Before starting 1 Please read through the entire protocol before proceeding A centrifuge with a deep well swing bucket rotor capable of holding a 96 well filter plate on top of a deep well culture plate is necessary for this protocol from Eppendorf or eguivalent Check Solution 2 for precipitate If present briefly warm solution at 37 C to re dissolve Prepare the complete Perfectprep BAC Solution 1 Perfectprep BAC Solution 1 plus RNAse A Solution and lyophilized Lysozyme Resuspend the lyophilized Lysozyme in the following amount of Perfectprep BAC Solution Kit Name Perfectprep BAC Perfectprep BAC Perfectprep BAC 96 96 Kit 2 Plts 96 Kit 10 Plts Base Kit 50 Lysozyme 40 mg 250 mg 2 x 625 mg lyophilized Add Perfectprep 1 8 ml 7ml 2x14 ml BAC Solution 1 Mix thoroughly by pipetting up and down or gently reversing the tube Take care to ensure that all of the powder is dissolved Some foaming will occur Pipet the entire content of the Lysozyme Solution to the
36. o long before applying the vacuum Verify that the cell pellets are completely thawed before adding Solution 1 to the culture plate Page 25 Manual Perfectprep BAC 96 Kit Low DNA yields Comments Suggestions Poor BAC DNA replication during host cell growth DNA molecules not resuspended in Elution Buffer prior to recovery by vacuum filtration or centrifugation Poorly grown cultures Frozen pellets were not completely thawed Some BAC clones do not replicate efficiently within their host cells Even though the yields may be low it is still possible to obtain adequate sequencing reads using the sequencing reaction conditions supplied in the protocol Ensure that the Elution Buffer is applied directly to the Filter Plate BAC membrane surface and not to the sides of the wells Make sure you incubate for 5 minutes Use the culture media and growth conditions given in Section 7 Verify that the cell pellets are completely thawed before adding Solution 1 to the culture plate Genomic DNA contamination Comments Suggestions Excessive cell lysis after addition of Solution 2 Poor DNA sequencing Mix gently by inverting the Culture Plate 5 times and do not exceed 5 minutes for the cell lysis incubation step Minor E coli genomic DNA contamination is possible but will not interfere with DNA sequencing Comments Suggestions Sub optimally grown cultures BAC template is d
37. on Lane Nottingham NG11 7EP Phone 44 1159 775 494 VWR International Hunter Boulevard Magna Park Lutterworth Leicestershire LE17 4XN Phone 44 77 694 624 42 E M ioMarke vwr com Web uk vwr com Contact person Becky Upton Hungaria VWR International o z Prievozska 6 821 09 Bratislava Slovakia Phone 4421 2 326 601 31 E Mail roman_kovacik vwr com Web www vwr com Contact person Roman Kovacik India Eppendorf India Limited Doshi Towers 4th Floor 156 Poonamallee High Road Kilpauk Chennai 600010 Phone 91 44 42 11 13 14 42 11 13 41 Fax 91 44 42 18 74 05 E Mail info eppendorf co in Web www eppendorf co in Ireland Isis Ballywaltrim Business Centre Boghall Road Bray Co Wicklow Phone 353 1 286 7777 Fax 353 1 286 7766 E mail enquiriesGisisco ie Web www isisco ie AGB Scientific Ltd a VWR International Company 136 Slaney Rd Dublin Industrial Estate Dublin 11 Phone 353 01 88 22 379 E Mail bioMarke vwr com Web http ie vwr com Contact person Ulrika Jansson Israel Zotal Ltd Habarzel 4 Tel Aviv 69710 Israel Phone 972 3 6492444 Fax 972 3 6496664 E Mail sales zotal co il Web www zotal co il Italy Eppendorf s r l Via Zante 14 20138 Milano Phone 39 02 55 4041 Fax 39 02 58 013 438 E Mail eppendorf eppendorf it Web www eppendorf it Latvia VWR International o z Prievozska 6 821 09 Bratislava Slovakia Phone 4421
38. or preparing the diluted Wash Buffer 95 100 isopropanol for preparing the diluted Trapping Buffer Molecular Biology Grade Water for use with the optional elution method see page 16 Incubator shaker at 37 C for bacterial culture growth in 96 well culture plates Centrifuge with microtiter plate rotor or deepwell plate rotor for pelleting bacterial cultures from Eppendorf or equivalent 8 channel electronic 1200 pl pipette from Eppendorf or equivalent 8 or 12 channel 10 100 ul manual pipette from Eppendorf or equivalent Absorbent material laboratory wipes benchtop protector or paper towel 200 additional plate seals for manual processing with the 50 plate kit only see ordering information on page 30 Air Permeable Seals when using the 50 plate and 50 plate base kits for growing bacterial cultures For vacuum protocol Vacuum manifold for processing 96 well filter bottom plates using the vacuum protocol such as 5 PRIME single basic manifold see page 28 Note The vacuum manifold must be compatible with the 96 well plates supplied in this kit Manifold manufacturers other than 5 PRIME have not been tested www 5Prime com Page 7 Manual Perfectprep BAC 96 Kit For centrifugation protocol 96 well catch plate capable of holding at least 2 ml for use with centrifugation protocol such as the 5 PRIME 2 4 ml Culture Plates See ordering information on page 30 Centrifuge with a deepwell plate rotor such as t
39. perature 14 Carefully remove the Plate Seal from Filter Plate BAC Slowly apply vacuum as performed in previous vacuum steps When all liquid has passed through bleed vacuum as before and allow chamber pressure to equalize to the outside 15 Add 600 pl diluted Wash Buffer to Filter Plate BAC Vacuum using above vacuum procedure 16 Remove Filter Plate BAC from the top of the vacuum manifold Thoroughly blot the bottom of the Filter Plate BAC several times on clean absorbent material Wipe excess liguid from bottom of Filter Plate BAC 17 Remove Culture Plate from the manifold and discard 18 Air dry Filter Plate BAC over the empty manifold chamber for 5 minutes Blot the bottom of the Filter Plate BAC several times on a clean absorbent material to eliminate residual diluted Wash Buffer Place a tall adapter in the manifold chamber Place a Collection Plate in the chamber on top of the adapter Page 36 www 5Prime com Manual Perfectprep BAC 96 Kit 19 Close the manifold lid Place Filter Plate BAC on top of the manifold in the same orientation as the Collection Plate 20 Add 30 pl of Elution Buffer to the center of each well of the Filter Plate BAC Incubate for 5 minutes at room temperature Vacuum liquid through the Filter Plate BAC using vacuum procedure previously described 21 Apply a second 30 ul aliquot of Elution Buffer to Filter Plate BAC 22 Vacuum as previously described 23 DNA is ready to use in downstream applications w
40. plications with the purified BAC DNA Inoculations should be performed using a 96 pin replicator or multi channel pipet using aseptic technique Glycerol stocks should be stored at 80 C and should not be allowed to thaw for more than one hour Repeated freeze thaw cycles of glycerol stocks should also be avoided to maintain the integrity of the cells Growth and storage of bacterial cultures 1 Pipet 1 5 ml of 2x YT bacterial growth medium supplemented with the appropriate antibiotic into each well of a Perfectprep 96 BAC Culture Plate 2 4 ml deep well culture plate See page 8f for information on antibiotics 2x YT medium is recommended for growth but other media may be used with this kit See page 8f www 5Prime com Page 9 Manual Perfectprep BAC 96 Kit e EEE 2 Inoculate each well containing growth medium with a single BAC clone from glycerol stock or a single colony See page 9 3 Seal the plate using an Air Permeable Seal to protect against cross well contamination while allowing the cultures to obtain sufficient aeration Secure plates in an incubator air shaker compatible with plate growth and incubate cultures for approximately 22 26 hours at 37 C while shaking at 325 rpm Page 10 www 5Prime com Manual Perfectprep BAC 96 Kit Vacuum protocol Before starting Please read through the entire protocol before proceeding Check Perfectprep BAC Solution 2 for precipitate If present briefly warm solution at 37 C
41. the Plate Seal Apply pressure to the lid with both hands and mix by inversion 4 times Caution While mixing it is extremely important to keep constant pressure against the Plate Seal across the entire plate to prevent leakage and cross well contamination 16 Incubate for 5 minutes at room temperature 17 Place the Filter Plate BAC over the Waste Plate from Step 12 Transfer the Catch Plate contents to the corresponding wells of the Filter Plate BAC 18 Centrifuge the Filter Plate BAC Waste Plate assembly at 1 900 x g for 2 minutes Check to make sure all of the cleared lysate has passed through the Filter Plate BAC If not centrifuge at 1 900 x g for an additional 2 minutes Page 20 www 5Prime com Manual Perfectprep BAC 96 Kit 19 20 21 22 23 24 25 26 21 28 The BAC DNA molecules are now trapped on the surface of the membrane in the Filter Plate BAC Add 600 ul of diluted Wash Buffer to each well of the Filter Plate BAC Centrifuge the Filter Plate BAC Waste Plate assembly at 1 900 x g for 2 minutes Check to make sure all of the liguid has passed through the Filter Plate BAC If not centrifuge at 1 900 x g for an additional 2 minutes Discard the flow through from Waste Plate Blot the bottom of the Filter Plate BAC several times on a clean absorbent material Wipe excess liguid from around the edges of the bottom of the Filter Plate BAC with an absorbent material Note Failure to blot t
42. ual Perfectprep BAC 96 Kit Troubleshooting Poor cell growth Comments Suggestions Use of wrong culture medium Use of wrong growth conditions Poor quality of glycerol stock Filter Plate A clogs Use 2x YT medium to achieve the maximum yield with the growth conditions outlined in the protocol If other media are used the growth time may have to be optimized Grow plates for 22 26 hours at 37 C and 325 rpm with proper aeration Optimal growth times vary between clones due to differences in the host strain and vector so test different times within the given range to determine what works best for the clones you are using Avoid repeated freeze thaw cycles and do not let glycerol stocks remain thawed for more than 1 hour Comments Suggestions Inadequate cell resuspension Un equalized pressure applied across the plate during vacuum filtration Precipitated material from alkaline lysate clogging the filter membrane Frozen pellets were not completely thawed www 5Prime com Ensure that cell pellets are completely resuspended in Solution 1 before lysing cells with Solution 2 Slowly apply the vacuum until it reaches the highest pressure and then apply a plate seal if some wells are still clogged Apply the lysate to the sides of the wells of the Filter Plate A so that they are not directly applied to the membrane surface Do not let the lysate sit on the Filter Plate A membrane for to
43. ww 5Prime com Page 37 Manual Perfectprep BAC 96 Kit Page 38 www 5Prime com Manual Perfectprep BAC 96 Kit www 5Prime com Page 39 Manual Perfectprep BAC 96 Kit 1044377 www 5Prime com
44. y sealed at room temperature www 5Prime com Page 11 Manual Perfectprep BAC 96 Kit Prepare appropriate amount of diluted Wash Buffer One plate Combine 22 5 ml of Wash Buffer Concentrate and 52 5 ml of 95 100 Ethanol in a new bottle Mix by inversion and store tightly sealed at room temperature Two plates Combine 45 ml of Wash Buffer Concentrate and 105 ml 95 100 Ethanol in a new bottle Mix by inversion and store tightly sealed at room temperature Prepare vacuum manifold Connect manifold to vacuum source using appropriate tubing The vacuum source must be able to create a vacuum of approximately 70 kPa or 20 inches of mercury Check to make sure that all valves are in the closed position and that bleed valves are in the open position Detailed vacuum protocol 1 Pellet bacteria by centrifuging the Culture Plate at 1 900 x g for 10 minutes 2 Aftercentrifugation pour off supernatants and blot any remaining medium from the plate by inverting and tapping the plate on a clean absorbent material Residual medium left in the wells can negatively affect the prep 3 Pelleted bacterial cells can be used immediately covered with a Plate Seal and stored at 4 C for up to four hours or 20 C for up to two months It is recommended to use bacterial cells immediately 4 Add 200 ul of Solution 1 to the cell pellets in each well of the Culture Plate Cover plate securely using a Plate Seal 5 Vortex the plate 2 3 minutes o
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