Protocols and Applications Guide (US Letter Size) - Promega

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||||||||| 9DNA Purification and HeLa, the amount of endotoxin present for routine transfections has a minimal effect on the efficiency of transfection (Butash et al. 2000). Many factors influence transfection efficiency and/or cellular death including the type and amount of transfection reagent, cell confluency, DNA amount and incubation time with the reagent:DNA complex. Each of these factors will need to be optimized for each cell line-plasmid combination transfected in order to minimize cell death and maximize transfection efficiency. In our experience, transfection experiments with HeLa and NIH/3T3 cells demonstrated that there was little DNA preparation difference with four different plasmid isolation systems used (based on silica membrane, anion exchange and silica resin) when comparing efficiencies using the same transfection reagent. However, the transfection reagent used for DNA uptake had a significant effect on transfection efficiency and cell death. For a starting place for optimization, visit the Transfection Calculator (www.promega.com /techserv/tools/transcalc/). To see if your cell line has been successfully transfected with Promega reagents, go to the Transfection Assistant (www.promega.com /transfectionasst/) for peer-reviewed citations and transfection information. Additional Resources about Endotoxin Online Tools eNotes FAQspeak: What methods exist to remove endotoxin contamination of plasmid DNA? (www.promega.com /enotes/faqspeak/0004/fq0021.htm) Protocols & Applications Guide www.promega.com rev. 3/09 IV. Plasmid DNA Purification Systems A. PureYield Plasmid Miniprep System The PureYield Plasmid Miniprep System (Cat.# A1221, A1222) isolates high-quality plasmid DNA for use in eukaryotic transfection and in vitro expression experiments. The unique reagents, proprietary matrix and silica membrane-based minicolumn design of the PureYield Plasmid Miniprep System allow rapid DNA purification directly from bacterial culture in less than 10 minutes with elution volumes as low as 30µl, resulting in more concentrated plasmid DNA. The low elution volume is possible because the column design retains virtually no buffer. While the unique Endotoxin Removal Wash removes protein, RNA and endotoxin contaminants from the bound DNA, the Column Wash Solution followed by membrane drying eliminates salts and alcohols from the plasmid prep, allowing use of the purified plasmid for highly sensitive applications such as transfection, in vitro transcription and coupled in vitro transcription/translation. An additional benefit is that the same degree of purification can be obtained even with low-copy-number plasmids. Although the system works best for plasmids less than 10kb, plasmids as large as 18kb have been purified. The unique combination of reagents in the PureYield Plasmid Miniprep System allows purification of plasmid either directly from bacterial culture or cell pellets of up to 3ml of cell culture (Figure 9.5). A typical overnight culture is grown in LB medium for 16–18 hours, and 0.6ml is used for the direct isolation method. If a larger volume is chosen, cells are harvested by centrifugation, then resuspended in 600µl of TE buffer or water. A transfection comparison of plasmid isolated using the PureYield Plasmid Miniprep in various cell lines can be found in Figure 9.6. PROTOCOLS & APPLICATIONS GUIDE 9-10
||||||||| 9DNA Purification PureYield Plasmid Miniprep: Centrifugation Protocol Lyse cells. Neutralize. Centrifuge. Transfer supernatant to PureYield Minicolumn. Place PureYield Minicolumn in a Collection Tube. Centrifuge. Add Endotoxin Removal Wash. Centrifuge. Add Column Wash Solution. Centrifuge. Transfer PureYield Minicolumn to a clean 1.5ml microcentrifuge tube. Add Elution Buffer or water. 1 minute 3 minutes 1 minute 30 seconds 30 seconds 30 seconds 1 minute Overnight culture Centrifuge to elute DNA. 1 minute 1 minute Centrifuge to elute DNA.
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CONTENTS I. Nucleic Acid Amplificat
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| 1Nucleic Acid Amplification CONTE
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| 1Nucleic Acid Amplification Unamp
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| 1Nucleic Acid Amplification Capoz
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| 1Nucleic Acid Amplification is co
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| 1Nucleic Acid Amplification One i
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| 1Nucleic Acid Amplification React
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| 1Nucleic Acid Amplification comig
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| 1Nucleic Acid Amplification inclu
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| 1Nucleic Acid Amplification polym
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| 1Nucleic Acid Amplification Addit
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| 1Nucleic Acid Amplification 3. Pl
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| 1Nucleic Acid Amplification 13. S
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| 1Nucleic Acid Amplification IX. R
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| 1Nucleic Acid Amplification Hoppe
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|| 2RNA Interference CONTENTS I. RN
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|| 2RNA Interference zebrafish (War
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|| 2RNA Interference Additional Res
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|| 2RNA Interference 2. Combine sep
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|| 2RNA Interference Target Site Se
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|| 2RNA Interference Transient Tran
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|| 2RNA Interference VII. Reference
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|| 2RNA Interference Wianny, F. and
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||| 3Apoptosis I. Introduction A. C
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||| 3Apoptosis ER stress pathway (H
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||| 3Apoptosis pathways and demonst
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||| 3Apoptosis Buffer Substrate Tha
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||| 3Apoptosis System, Fluorometric
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||| 3Apoptosis Qi, H. et al. (2003)
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||| 3Apoptosis 2. Deparaffinize by
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||| 3Apoptosis • 0.1% Triton® X-
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||| 3Apoptosis 2. Pre-equilibrate t
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||| 3Apoptosis Fluorescence (560/59
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||| 3Apoptosis Ellis, H.M. and Horv
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|||| 4Cell Viability CONTENTS I. In
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|||| 4Cell Viability Table 4.1. Com
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|||| 4Cell Viability E. Homogeneous
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|||| 4Cell Viability equilibration
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|||| 4Cell Viability 5. Optional: I
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|||| 4Cell Viability Online Tools C
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|||| 4Cell Viability 5. Shake gentl
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|||| 4Cell Viability Crouch, S.P.M.
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||||| 5Protein Expression I. Introd
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||||| 5Protein Expression B. DNA-Dr
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||||| 5Protein Expression Figure 5.
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|||||| 6Expression Analysis I. Intr
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Page 133 and 134: ||||||| 7Cell Signaling CONTENTS I.
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Page 209 and 210: |||||||||| 10Cell Imaging I. Introd
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|||||||||||| 12Transfection I. Intr
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||||||||||||| 13Cloning CONTENTS I.
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||||||||||||| 13Cloning 1257bp PCR
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||||||||||||| 13Cloning Promega Pub
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||||||||||||| 13Cloning Ligation 1.
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||||||||||||| 13Cloning 7. Recommen
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||||||||||||| 13Cloning Figure 13.9
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|||||||||||||| 14DNA-Based Human Id
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Protocols and Applications Guide (US Letter Size) - Promega

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