CalPhos Mammalian Transfection Kit User Manual
Contents
1. malian Transfection Kit You may want to try incubation times from 1 18 hours for optimization After transfections have been optimized scale up or scale down as necessary for the size of culture plate you are using see Appendix B for a table of conversions Clontech Laboratories Inc www clontech com Protocol No PT3025 1 8 Version No PR732205 CalPhos Mammalian Transfection Kit User Manual Appendix B Culture Plate Conversions TABLE I CULTURE PLATE CONVERSION Size of Plate Growth Area Relative Area Recommended cm2 Volume 96 well 0 32 0 04 X 200 ul 24 well 1 88 0 25 X 500 ul 12 well 3 83 0 5 X 1 0 ml 6 well 9 4 12 X 2 0 ml 35mm 8 0 10 X 2 0 ml 60 mm 21 26 X 5 0 ml 10 cm 55 7 X 10 0 ml Flasks 25 3 X 5 0 ml 75 9 X 12 0 ml Relative area is expressed as a factor of the growth area of a 35 mm culture plate Notice to Purchaser This product is intended to be used for research purposes only It is not to be used for drug or diagnostic purposes nor is it intended for human use Clontech products may not be resold modified for resale or used to manufacture commercial products without written approval of Clontech Laboratories Inc NucleoBond anion exchange resin is covered under European Patent No 496822 Nucleo Bond is a registered trademark of MACHEREY NAGEL GmbH amp Co KG Clontech Clontech logo and all other trademarks are the property of Clontech Laboratories Inc unless noted otherwise Cl2. Sons Inc NY Vol 1 Ch 9 Freshney R I 2000 Culture of Animal Cells Fourth Edition Wiley Liss NY Clontech Laboratories Inc www clontech com Protocol No PT3025 1 6 Version No PR732205 CalPhos Mammalian Transfection Kit User Manual VII Related Products For the latest and most complete listing of all Clontech products please visit www clontech com Cat No e Clonfectin 631301 e Living Colors Fluorescent Protein many Reporter Vectors e Tet On and Tet Off Expression many Systems and Vectors e Great EscAPe Secreted Alkaline many Phosphatase SEAP Vectors and Kits e galactosidase Vectors and Kits many e IRES Expression Vectors many e Retro X Expression Vectors many e MATCHMAKER Mammalian Two Hybrid 630301 Assay Kit e NucleoBond6 Plasmid Kits Mini Kit 635925 635926 635927 Midi Kit 635929 635930 635931 Maxi Kit 635933 635934 635935 635936 Mega Kit 635938 Giga Kit 635939 e NucleoBond AXTips many Protocol No PT3025 1 www clontech com Clontech Laboratories Inc Version No PR732205 7 CalPhos Mammalian Transfection Kit User Manual Appendix A Optimization of Transfection The efficiency of a mammalian cell transfection is primarily dependent on the host cell line used Optimization of the transfection parameters for each cell type is crucial to obtaining consistently successful transfections There fore for each cell type you plan to use perform preliminary expe
3. N NaOH Add deionized H O to final volume of 2 L Store at room temperature e 1XTrypsin EDTA Life Technologies Cat No 25300 054 e Plasmid DNA The DNA should be of high quality e g double CsCl banded or column purified DNA Clontech offers many NucleoBond Plasmid Purification Kits and cartridges which yield transfection grade plasmids See Related Products for more information Clontech Laboratories Inc www clontech com Protocol No PT3025 1 4 Version No PR732205 CalPhos Mammalian Transfection Kit User Manual IV CalPhos Mammalian Transfection Protocol The following protocol is designed for use with adherent cultures growing in 35 mm tissue culture plates If you are using plates wells or flasks of a different size adjust the volume of the transfection solution in accordance with your culture volume See Appendix B for culture plate conversions All steps of the following protocol should be performed in a sterile tissue culture hood 1 10 11 Plate the cells the day before the transfection experiment The cells should be 50 80 confluent the day of transfection Generally we plate 4 x 10 cells 35 mm plate 0 5 3 hr prior to transfection replace culture medium on plates to be transfected with 2 ml of fresh culture medium per 35 mm plate For each transfection prepare Solution A and Solution B in separate sterile tubes Solution A add components in the following order 2 4 ug Pl
4. transfection solution Incubate 20 min e Apply transfection solution to subconfluent cell culture with complete growth medium Assay for transient gene expression or Begin selection for stable transformants 24 72 hr post transfection Figure 1 Flow chart for using the CalPhos Mammalian Transfection Kit Protocol No PT3025 1 www clontech com Clontech Laboratories Inc Version No PR732205 CalPhos Mammalian Transfection Kit User Manual ll List of Components Store HBS at 20 C store all other components at 4 C The following reagents are sufficient for 100 transfections in 10 cm plates or 725 transfections in 35 mm plates 9ml 2 M Calcium Solution 2x35ml 2X HEPES Buffered Saline HBS We recommend dispensing this buffer into small aliquots to be stored at 209C Avoid multiple freeze thaw cycles When an aliquot is in use store it at 4 C for up to one week 2x 35 ml Sterile H O Ill Additional Materials Required e Cell culture plates or flasks e Tubes 12 x 75 mm sterile tubes e Cell culture medium appropriate growth medium for mammalian cells in culture e Fetal bovine serum newborn calf serum or equivalent to supplement the growth medium e Phosphate buffered saline PBS pH 7 4 Final Conc To prepare 2 L of solution Na HPO 58 mM 16 5 g NaH PO 17 mM 4 1 g NaCl 68 mM 8 0 g Dissolve the above components in 1 8 L of deionized H O Adjust to pH 7 4 with 0 1
5. CalPhos Mammalian Transfection Kit User Manual Cat No 631312 PT3025 1 PR732205 Published 28 March 2007 CalPhos Mammalian Transfection Kit User Manual Table of Contents ll lll IV V VI VII Introduction List of Components Additional Materials Required CalPhos Mammalian Transfection Protocol Troubleshooting Guide References Related Products Appendix A Optimization of Transfection Appendix B Culture Plate Conversions Clontech Laboratories Inc www clontech com o o 3 o o Ph AOG Protocol No PT3025 1 Version No PR732205 CalPhos Mammalian Transfection Kit User Manual Introduction The ability to introduce exogenous DNA into cultured cells is a powerful tool for molecular and cell biologists Of the many methods to introduce DNA into mammalian cell cultures the calcium phosphate method is one of the most widely used because it is inexpensive simple and suitable for a range of different cell types Ausubel et al 1994 Graham amp van der Eb 1973 The CalPhos Mammalian Transfection Kit provides high quality pretested reagents suitable for both transient and stable transfections The kit includes all the reagents necessary to perform 100 transfections in 10 cm plates or 725 transfections in 35 mm plates Solution A Solution B Plasmid DNA 2X HBS Calcium solution Add Solution A to Solution B while vortexing ea eS e Sa SE Incubate 2 12 hr Replace
6. asmid DNA Sterile H O 12 4 ul 2 M Calcium Solution 100 ul Total Volume Solution B 100 ul 2X HBS Note To reduce variability when transfecting multiple plates with the same plasmid DNA prepare master solutions of Solutions A and B sufficient for all plates Carefully and slowly vortex Solution B while adding Solution A dropwise Alternatively blow bubbles into Solution B with a 1 ml sterile pipette and an autopipettor while adding Solution A drop wise Incubate the transfection solution at room temperature for 20 min Gently vortex transfection solution and then add solution dropwise to culture plate medium Add 200 ul of transfection solution per 35 mm plate Gently move plates back and forth to distribute transfection solution evenly Do not rotate plates as this will concentrate transfection precipitate in the center of the well or plate Incubate plates at 37 C for 2 12 hr in a CO incubator Remove calcium phosphate containing medium and wash cells with medium or 1X PBS Feed plate with 2 ml fresh complete growth medium and incubate at 37 C until needed for assay Assay for transient gene expression or start selection for stable transformants 24 72 hr post transfection Protocol No PT3025 1 www clontech com Clontech Laboratories Inc Version No PR732205 5 CalPhos Mammalian Transfection Kit User Manual V Troubleshooting Guide A Low Transfection Efficiency e Poor precipita
7. ontech is a Takara Bio Company 2007 Protocol No PT3025 1 www clontech com Clontech Laboratories Inc Version No PR732205 9
8. riments to determine the optimal 1 cell density 2 amount and purity of DNA and 3 transfection incubation time For the preliminary experiments the host cell line can be transfected with a reporter expression vector such as pCMVB Cat No 631719 or pSEAP2 Control Vector Cat No 631717 The success of the transfection can then be estimated by assaying for B galactosidase or secreted alkaline phospha tase Once the transfection parameters have been optimized they should be kept consistent from one experiment to the next to obtain reproducible results The following is a general guideline for optimizing the transfection param eters To optimize transfection parameters it is best to perform a series of small scale transfections This can be done conveniently in 12 well or 6 well plates To optimize cell density keeping all other parameters constant plate hostcells in individual wells of a 6 well plate at varying densities e g 5 x 104 1 x 10 2 x 10 4 x 10 8 x 105 24 72 hours post transfection assay for reporter gene SEAP or galactosidase activity Record results Repeat the experi ment once or twice to account for day to day variation Choose the density with the highest reporter gene activity The other parameters can be optimized in much the same way Hold all other variables constant while varying the parameter you are testing Transfection incubations should be maximal at 2 16 hours using the CalPhos Mam
9. te formation Solution Addition of the calcium DNA Solution A to the 2X HBS Solution B should be performed dropwise and with continuous mixing Adding Solution A too quickly or with too little mixing can result in a poor precipitate e Poor quality DNA Solution The A o Ag9 ratio of the plasmid DNA should be gt 1 7 pH not optimal Solution The pH of the HBS should be between 7 05 and 7 12 How ever during prolonged storage the pH of the solution may change therefore use the transfection kit within the shelf life indicated on the accompanying Product Analysis Certificate PAC B Variable Transfection Efficiency in Experiments There will always be some variability in transfection efficiencies We recommend performing transfections in triplicate to minimize the vari ability e Variable cell density Solution Keep cell density constant after optimizing transfection procedures Generally we use cultures that are 50 80 confluent at the time of transfection e Suboptimal cell growth Solution Keep cells healthy in culture Cells should be in mid log phase growth when plated for transfection Transfection efficien cies may decrease for cell lines that have been passaged for too many generations VI References Ausubel F M Brent R Kingston R E Moore D D Seidman J G Smith J A amp Struhl K 1994 In Current Protocols in Molecular Biology Greene Publishing Associates and John Wiley amp
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