Protocols and Applications Guide (US Letter Size) - Promega

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||||| 5Protein Expression µl membranes precursor> (~31.5kDa) processed> (~28.9kDa) 0371TA mRNA β-lactamase α-factor 0 0.3 0.6 0.9 1.2 1.5 1.8 2.1 2.4 0 0.3 0.6 0.9 1.2 1.5 1.8 2.1 2.4 Signal Processing Glycosylation
||||| 5Protein Expression Circular System requires that the gene be under the control of either a T7 or a good E. coli promoter. Expression levels from T7 promoters are typically higher than that from E. coli promoters in this extract. Expression from E. coli promoters can be inhibited by the addition of rifampicin to the extract; transcription by T7 RNA Polymerase is resistant to rifampicin. S30 Extract for Linear DNA or RNA Expression of gene products from linear DNA containing supercoiling-sensitive promoters can be reduced in the S30 System by up to 100-fold (Chen and Zubay, 1983). Examples of good promoters that are supercoiling-insensitive include lacUV5, tac, λ and λPR. DNA from other prokaryotic species may not contain promoters that direct transcription in the E. coli S30 Extract System for Linear Templates. RNA generated in vitro from cloned genes lacking an E. coli promoter is also suitable. Larger templates, such as bacteriophage lambda DNA, may also be used. PCR-Generated Templates PCR technology has introduced many methods for site-specific in vitro mutagenesis. Combining PCR with phage λ exonuclease treatments has produced mutated fragments larger than 2.5kb (Shyamala and Ames, 1991). PCR products can be added to the E. coli S30 Extract System for Linear Templates for rapid confirmation of expected protein size or activity. Avoid contaminating the S30 Extract reaction with the wrong PCR product or primer dimers. If agarose gel analysis indicates that your PCR reaction produced a unique band, primer dimers can be removed by ethanol precipitation with sodium acetate. Otherwise, PCR-amplified DNA should be gel purified before use. Restriction Enzyme-Digested Templates For restriction enzyme-digested DNA, 10–20µg of DNA should be digested in a 100–200µl reaction volume. Ethanol precipitate and resuspend the DNA at a concentration of 1µg/µl in TE buffer or water. Add 2–4µg of this DNA directly to the S30 reaction. However, if the desired results are not obtained, the DNA should be further purified by phenol extraction followed by ethanol precipitation. RNA Templates The amount of in vitro RNA added to the extract can vary from 10–100µg. For synthesizing milligram quantities of highly pure, “translatable” RNA, we recommend using one of the RiboMAX Large Scale RNA Production Systems (Cat.# P1280 and P1300; RiboMAX Large Scale RNA Production Systems—SP6 and T7 Technical Bulletin, #TB166 (www.promega.com/tbs/tb166/tb166.html). C. S30 T7 High-Yield Protein Expression System The S30 T7 High-Yield Protein Expression System is an E. coli extract-based cell-free protein synthesis system. It simplifies the transcription and translation of DNA sequences cloned in plasmid or lambda vectors containing a T7 promoter by providing an extract that contains T7 RNA polymerase for transcription and all necessary components for translation. This system can produce high Protocols & Applications Guide www.promega.com rev. 6/09 levels of recombinant proteins (up to hundreds of micrograms of recombinant protein per milliliter of reaction) within an hour using a vector containing the sequence of interest, a T7 promoter and a ribosome-binding site (RBS). The S30 T7 High-Yield Protein Expression System contains the T7 S30 Extract,Circular, which is prepared by modifications of the method described by Zubay (Zubay, 1973 and Zubay, 1980) from an E. coli strain B deficient in OmpT endoproteinase and lon protease activity. This results in greater stability for translated proteins that would otherwise be degraded by proteases if expressed in vivo (Studier and Moffatt, 1986; Pratt, 1984). An optimized S30 Premix Plus provides all other required components, including amino acids, rNTPs, tRNAs, an ATP-regenerating system, IPTG and appropriate salts to express high levels of recombinant proteins. Using this system, protein can be expressed in volumes as small as 5µl for high-throughput screening in a 96-well plate format. The amount of protein synthesized increases proportionally with reaction volume (up to 250µl). Because it is a cell-free system, the S30 T7 High-Yield Protein Expression System can be used to express proteins that are toxic to E. coli cells. Finally, small amounts of target protein can be purified using affinity tags (e.g., metal-affinity tag) for downstream analysis. For a detailed protocol and background information about the S30 T7 High-Yield Protein Expression System, please see Technical Manual #TM306 (www.promega.com/tbs/tm306/tm306.html). D. E. coli S30 Extract Systems Protocol Materials Required: • appropriate E. coli S30 Extract System (Cat.# L1020, L1030 or L1130) • radiolabeled amino acid • Nuclease-Free Water (Cat.# P1193) The following protocol is designed for use with the E. coli S30 Extract Systems for Circular (including the T7 System) or Linear DNA Templates and [35S]methionine as the radiolabel. Other radioisotopes may also be used (see Table 5.3). For positive control reactions, use 4µl of the Control DNA provided. For multiple reactions, create a master mix by combining the appropriate volumes of Amino Acid Mixture Minus Methionine (or Cysteine or Leucine), S30 Premix Without Amino Acids, radiolabeled amino acid (optional), S30 Extract and Nuclease-Free Water. Aliquot the master mix into 1.5ml microcentrifuge tubes and initiate the reactions by adding the DNA template to the tubes. PROTOCOLS & APPLICATIONS GUIDE 5-15
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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 VII. Reference
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|| 2RNA Interference Wianny, F. and
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||| 3Apoptosis I. Introduction A. C
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Page 69 and 70: |||| 4Cell Viability CONTENTS I. In
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Page 133 and 134: ||||||| 7Cell Signaling CONTENTS I.
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Table 8.1. pGL4 Luciferase Reporter
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||||||||| 9DNA Purification I. Intr
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|||||||||| 10Cell Imaging I. Introd
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||||||||||||| 13Cloning CONTENTS I.
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