Protocols and Applications Guide (US Letter Size) - Promega

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||||||||| 9DNA Purification tube defect (NTD) risk factor within the Irish population. To survey for common variations in MTHFR, genomic DNA was extracted from blood, and exons 1–11 of MTHFR were amplified and sequenced with BigDye® Terminator mix. The Wizard® MagneSil® Sequencing Reaction Clean-Up System was used to purify the sequencing reactions prior to analysis on an ABI PRISM® 377 DNA sequencer. PubMed Number: 16145688 X. Fragment/PCR Product Purification Protocol Featuring the Wizard® SV Gel and PCR Clean-Up System Materials Required: • Wizard® SV Gel and PCR Clean-Up System (Cat.# A9281) • 1.5ml microcentrifuge tubes • ethanol (95%) • Vacuum Adapters (Cat.# A1331; only for vacuum purification) • agarose gel (standard or low-melting point; only for gel purification) • 1X TAE or TBE electrophoresis buffer (only for gel purification) • 50–65°C heating block (only for gel purification) A. Preparing the Membrane Wash Solution Add the indicated volume of 95% ethanol to the Membrane Wash Solution prior to beginning the procedure (see Table 9.11). Mark the bottle label to record that this addition was made. Tightly close the bottle cap after each use to prevent evaporation. Table 9.11. Volume of 95% Ethanol to Add to Membrane Wash Solution for Each System Size. System Size Volume of 95% Ethanol 10 preps 15ml 50 preps 75ml 250 preps 375ml B. DNA Purification by Centrifugation Gel Slice and PCR Product Preparation Dissolving the Gel Slice 1. Following electrophoresis, excise DNA band from gel and place gel slice in a 1.5ml microcentrifuge tube. 2. Add 10µl of Membrane Binding Solution per 10mg of gel slice. Vortex and incubate at 50–65°C until gel slice is completely dissolved. Processing PCR Amplifications 1. Add an equal volume of Membrane Binding Solution to the PCR amplification. Protocols & Applications Guide www.promega.com rev. 3/09 Binding of DNA 1. Insert SV Minicolumn into Collection Tube. 2. Transfer dissolved gel mixture or prepared PCR product to the Minicolumn assembly. Incubate at room temperature for 1 minute. 3. Centrifuge at 16,000 × g for 1 minute. Discard flowthrough and reinsert Minicolumn into Collection Tube. Note: If the volume of the DNA solution is >700µl, repeat Steps 2 and 3, transferring ≤700µl until all the solution has been processed. Washing 4. Add 700µl of Membrane Wash Solution (ethanol added). Centrifuge at 16,000 × g for 1 minute. Discard flowthrough and reinsert Minicolumn into Collection Tube. 5. Repeat Step 4 with 500µl of Membrane Wash Solution. Centrifuge at 16,000 × g for 5 minutes. 6. Empty the Collection Tube and recentrifuge the column assembly for 1 minute with the microcentrifuge lid open (or off) to allow evaporation of any residual ethanol. Elution 7. Carefully transfer Minicolumn to a clean 1.5ml microcentrifuge tube. 8. Add 50µl of Nuclease-Free Water to the Minicolumn. Incubate at room temperature for 1 minute. Centrifuge at 16,000 × g for 1 minute. 9. Discard Minicolumn and store DNA at 4°C or –20°C. C. DNA Purification by Vacuum Gel Slice and PCR Product Preparation Dissolving the Gel Slice 1. Following electrophoresis, excise DNA band from gel and place gel slice in a 1.5ml microcentrifuge tube. 2. Add 10µl of Membrane Binding Solution per 10mg of gel slice. Vortex and incubate at 50–65°C until gel slice is completely dissolved. Processing PCR Amplifications 1. Add an equal volume of Membrane Binding Solution to the PCR amplification. Binding of DNA 1. Attach Vacuum Adapter to manifold port and insert SV Minicolumn into Adapter. 2. Transfer dissolved gel mixture or prepared PCR product to the Minicolumn. Incubate at room temperature for 1 minute. PROTOCOLS & APPLICATIONS GUIDE 9-34
||||||||| 9DNA Purification 3. Apply vacuum to pull liquid through Minicolumn. Release vacuum when all liquid has passed through Minicolumn. Washing 4. Add 700µl of Membrane Wash Solution (ethanol added). Apply a vacuum to pull solution through Minicolumn. 5. Turn off vacuum and repeat Step 4 with 500µl of Membrane Wash Solution. Apply a vacuum to pull solution through Minicolumn. 6. Transfer Minicolumn to a Collection Tube. Centrifuge at 16,000 × g for 5 minutes. 7. Empty the Collection Tube and recentrifuge the column assembly for 1 minute with the microcentrifuge lid open (or off) to allow evaporation of any residual ethanol. Elution 8. Carefully transfer Minicolumn to a clean 1.5ml microcentrifuge tube. 9. Add 50µl of Nuclease-Free Water to the Minicolumn. Incubate at room temperature for 1 minute. Centrifuge at 16,000 × g for 1 minute. 10. Discard Minicolumn and store DNA at 4°C or –20°C. XI. Composition of Solutions LB (Luria-Bertani) medium (1 liter) 10g Bacto®-tryptone 5g Bacto®-yeast extract 5g NaCl Adjust pH to 7.5 with NaOH. Autoclave. LB-Miller medium (1 liter) 10g Bacto®-tryptone 5g Bacto®-yeast extract 10g NaCl Adjust pH to 7.0 with NaOH. Autoclave. Membrane Wash Solution (Wizard® SV Gel and PCR Clean-Up System) 10mM potassium acetate (pH 5.0) 80% ethanol (after ethanol addition) 16.7µM EDTA (pH 8.0) To prepare this solution, add 95% ethanol to the supplied Membrane Wash Solution (concentrated) as described in Table 9.11 in the Fragment/PCR Product Purification protocol section. Protocols & Applications Guide www.promega.com rev. 3/09 Membrane Binding Solution (Wizard® SV Gel and PCR Clean-Up System) 4.5M guanidine isothiocyanate 0.5M potassium acetate (pH 5.0) 1X TE buffer 10mM Tris-HCl (pH 7.5) 1mM EDTA (pH 8.0) 1X TBE buffer 89mM Tris base 89mM boric acid 2mM EDTA (pH 8.0) 1X TAE buffer 40mM Tris base 5mM sodium acetate 1mM EDTA (pH 8.0) Terrific Broth (1 liter) 12g Bacto®-tryptone 24g Bacto®-yeast extract 4ml glycerol Add components to 900ml deionized water. Autoclave and allow solution to cool to ~60°C. Add 100ml of a sterile solution of 0.17M KH2PO4, 0.72M K2HPO4 and mix to disperse evenly. 0.17M KH2PO4, 0.72M K2HPO4 sterile solution 2.31g KH2PO4 12.54g K2HPO4 Dissolve in 90ml deionized water. Adjust volume to 100ml and sterilize by autoclaving. YPD broth (1 liter) 10g yeast extract 20g peptone 20g dextrose Autoclave. Final pH 6.5±0.2 at 25°C. 2X YT medium (1 liter) 16g Bacto®-tryptone 10g Bacto®-yeast extract 5g NaCl Adjust pH to 7.0 with NaOH. Autoclave. XII.References Adams, D.S. (2003) In: Lab Math: A Handbook of Measurements, Calculations, and Other Quantitative Skills for Use at the Bench Chapter 5, Cold Spring Harbor Laboratory Press, NY, 127–45. Ahmed, A. et al. (2003) Madurella mycetomatis strains from mycetoma lesions in Sudanese patients are clonal. J. Clin. Microbiol. 41, 4537–41. Ausubel, F.M. et al. (1989) Current Protocols in Molecular Biology, Vol. 2, John Wiley and Sons, NY. Birnboim, H.C. (1983) A rapid alkaline extraction method for the isolation of plasmid DNA. Methods Enzymol. 100, 243–55. Birnboim, H.C. and Doly, J. (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucl. Acids Res. 7, 1513–23. PROTOCOLS & APPLICATIONS GUIDE 9-35
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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 Qi, H. et al. (2003)
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||| 3Apoptosis Fluorescence (560/59
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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 Online Tools C
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||||| 5Protein Expression I. Introd
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||||| 5Protein Expression Figure 5.
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|||||| 6Expression Analysis I. Intr
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||||||| 7Cell Signaling CONTENTS I.
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||||||| 7Cell Signaling SMALL GTP-B
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||||||| 7Cell Signaling HO S N N S
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||||||| 7Cell Signaling PN083 Intro
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||||||| 7Cell Signaling (continued
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|||||||||||| 12Transfection I. Intr
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||||||||||||| 13Cloning CONTENTS I.
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||||||||||||| 13Cloning Figure 13.9
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