Protocols and Applications Guide (US Letter Size) - Promega

More documents

Recommendations

Info

|||||| 6Expression Analysis Choosing an RNA Isolation Protocol One of the first considerations when deciding on an RNA purification protocol is whether you will be using total RNA or poly(A)+ RNA for your application. The source of RNA, type of RNA to be purified, relative abundance of the RNA, sample size and convenience of the isolation procedure are all factors that also must be considered. For valuable tissue samples, we suggest that a portion of each sample be reserved at –70°C in the event that loss of a sample occurs during RNA purification. Promega offers both total RNA isolation systems (SV Total RNA Isolation System, PureYield RNA Midiprep System and MagneSil® Total RNA mini-Isolation System) and poly(A)+ RNA isolation systems (PolyATtract® System 1000) that yield clean, intact RNA from a variety of cell and tissue types. Additional Resources for RNA Isolation Technical Bulletins and Manuals TM048 SV Total RNA Isolation System Technical Manual (www.promega.com /tbs/tm048/tm048.html) TB294 SV 96 Total RNA Isolation System Technical Bulletin (www.promega.com/tbs/tb294/tb294.html) TM021 PolyATtract® mRNA Isolation Systems Technical Manual (www.promega.com /tbs/tm021/tm021.html) TM228 PolyATtract® System 1000 Technical Manual (www.promega.com /tbs/tm228/tm228.html) Promega Publications PN099 RNA purification kit comparison: Yield, quality and real-time RT-PCR performance (www.promega.com /pnotes/99/15674_12/15674_12.html) PN094 Cleanup of TRIzol® reagent-purified total RNA using the PureYield RNA Midiprep System (www.promega.com /pnotes/94/14410_07/14410_07.html) PN092 PureYield RNA Midiprep System: Isolating pure total RNA without DNase (www.promega.com /pnotes/92/13408_14/13408_14.html) PN086 MagneSil® Total RNA mini-Isolation System (www.promega.com /pnotes/86/11217_18/11217_18.html) PN084 Quantitative, real-time RT-PCR expression using the SV 96 Total RNA Isolation System (www.promega.com /pnotes/84/10705_23/10705_23.html) Protocols & Applications Guide www.promega.com rev. 10/08 PN079 High-throughput purification using the SV 96 Total RNA Isolation System (www.promega.com /pnotes/79/9492_29/9492_29.html) PN068 Isolation of RNA from plant, yeast and bacteria (www.promega.com /pnotes/68/7381_28/7381_28.html) PN064 RNA purification: A rapid and versatile protocol for the isolation of total RNA (www.promega.com /pnotes/64/9331_07/promega.html) PN063 Technically speaking: Tips for working with RNA and troubleshooting downstream applications (www.promega.com /pnotes/63/8581_17/promega.html) PN055 A rapid protocol for the isolation of total RNA suitable for RT-PCR (www.promega.com /pnotes/55/5265e/5265e.html) PN044 Isolation of mRNA from plant tissues using the PolyATtract® System 1000 (www.promega.com /pnotes/44/neille/neille.html) PN039 30-Minute poly(A)+ RNA isolation directly from tissue (www.promega.com /pnotes/39/39_07/39_07.htm) BR120 RNA Analysis Notebook (www.promega.com /guides/rna_guide/purifyingrna.pdf) eNotes Measuring gene expression from mammalian brain tissue (www.promega.com /enotes/applications/ap0007_tabs.htm) Total RNA Isolation Using the SV Total RNA Isolation System The SV Total RNA Isolation System provides a fast and simple technique to purify intact total RNA from tissues, cultured cells and white blood cells in as little as one hour, depending on the number of samples to be processed. Up to 60mg of tissue can be processed per purification, depending on the type and RNA expression levels of the tissue. The system also incorporates a DNase treatment step to substantially reduce genomic DNA contamination, which can interfere with amplification-based methods. For best results from this system, use fresh samples when processing tissue. Older samples may yield less total RNA. If necessary freeze the samples immediately after collection in liquid nitrogen, and store at –70°C for future use. Samples homogenized in SV RNA Lysis Buffer also may be stored at –70°C. PROTOCOLS & APPLICATIONS GUIDE 6-10
|||||| 6Expression Analysis For a protocol for RNA isolation from cultured cells, tissue samples, white blood cells, plant tissue, yeast and bacterial cells, see the SV Total RNA Isolation System Technical Manual #TM048 (www.promega.com/tbs/tm048/tm048.html). Materials Required: (see Composition of Solutions section) • SV Total RNA Isolation System (Cat.# Z3100, Z3101 or Z3105) and protocol • small tissue homogenizer (for RNA isolation from tissue) • ethanol, 95%, RNase-free • microcentrifuge • 10X phosphate-buffered saline (PBS), sterile (for RNA isolation from cultured cells) • sterile hypodermic syringe fitted with a sterile 20-gauge needle (for RNA isolation from cultured cells) • water bath or heating block, preheated to 70°C • Laboratory Vacuum Manifold (e.g., Vac-Man®, Cat.# A7231, or Vac-Man® Jr. Laboratory Vacuum Manifold, Cat.# A7660) and Vacuum Adapters (Cat.# A1331) (required for RNA purification by vacuum) Poly(A)+ RNA Isolation Using the PolyATtract® System 1000 The PolyATtract® System 1000 isolates poly(A)+ RNA directly from crude cell or tissue lysates using the Promega MagneSphere® technology, eliminating the need for oligo(dT) cellulose columns. The system uses a biotinylated oligo(dT) primer to hybridize in solution to the 3′ poly(A) region present in most mature eukaryotic mRNA species. The hybrids are captured using streptavidin coupled to paramagnetic particles and a magnetic separation stand, then washed at high stringency. The mRNA is eluted from the solid phase by the simple addition of RNase-free deionized water. This procedure yields an essentially pure fraction of mature mRNA after only a single round of magnetic separation. To isolate poly(A)+ RNA directly from tissue samples or cultured cells, see the PolyATtract® System 1000 Technical Manual #TM228 (www.promega.com /tbs/tm228/tm228.html). This technical manual also describes precipitation and concentration of mRNA and determination of mRNA concentration. Materials Required: (see Composition of Solutions section) • PolyATtract® System 1000 (Cat.# Z5400 or Z5420) and protocol • small tissue homogenizer (for RNA isolation from tissue) • 50ml sterile screw-cap conical tubes • 15ml sterile COREX® or other glass centrifuge tubes • 70°C water bath • Beckman Model J2-21 centrifuge or equivalent • 1X PBS (for RNA isolation from cell cultures) • scale or balance (to weigh tissue samples) • MagneSphere® Magnetic Separation Stand (see Table 1 of the PolyATtract® System 1000 Technical Manual Protocols & Applications Guide www.promega.com rev. 10/08 #TM228 (www.promega.com/tbs/tm228/tm228.html) to determine the appropriate magnetic stand) B. DNA and RNA Labeling A number of methods exist to attach a label to a nucleic acid molecule. These consist of techniques to incorporate the label into the substrate or attach the label to the ends of a nucleic acid fragment. The choice of method is determined largely by the nature of the substrate to be labeled. Some other factors to consider include: the amount of substrate available for labeling, its size in base pairs, the type of nucleic acid (DNA or RNA), the desired specific activity and whether it is double-stranded or single-stranded. Promega provides several nucleic acid-labeling systems, which are described briefly in this section. Random-Primed Labeling Random-primed labeling (Feinberg and Vogelstein, 1983; Feinberg and Vogelstein, 1984) uses a mixture of random hexadeoxyribonucleotides to prime DNA synthesis in vitro from any linear double-stranded DNA template. With this method, it is possible to generate probes of high specific activity (>1 × 109cpm/μg), even using DNA fragments cut from agarose gels (Feinberg and Vogelstein, 1984). Since the input DNA serves as a template and remains intact during the reaction, minimal amounts of DNA (25ng) can be labeled to a high specific activity. Using the Prime-a-Gene® Labeling System, 40–80% of the labeled deoxyribonucleotide can typically be incorporated into the DNA template, depending on the template and reaction conditions used. Using a template greater than 500bp, probes generated by random-primed labeling generally are 250–300bp in length and are suitable for a variety of applications, including Northern analysis. 5′ End Labeling 5′ end labeling uses T4 polynucleotide kinase, which catalyzes the transfer of the γ-phosphate group from ATP to the 5′-hydroxyl terminus of double-stranded or single-stranded DNA or RNA molecules (the forward reaction). Suitable substrates include synthetic oligonucleotides, most of which lack a 5′-phosphate group, and DNA fragments that have been dephosphorylated with alkaline phosphatase to remove the 5′-phosphate groups. Under certain conditions, the reaction with T4 polynucleotide kinase can be made reversible, permitting exchange of the γ-phosphate of ATP with the 5′ terminal phosphate of a polynucleotide (the exchange reaction, see the T4 Polynucleotide Kinase Technical Bulletin #TB519 (www.promega.com/tbs/tb519/tb519.html)), thus circumventing the need to dephosphorylate the substrate (Donis-Keller et al. 1977). The specific activity of a probe generated using the forward reaction is typically 2 × 106cpm/pmol, while the specific activity of a probe generated using the exchange reaction is approximately 6 × 105cpm/pmol (Berger and Kimmel, 1987). The Promega 5′ End-Labeling System includes both T4 Polynucleotide Kinase and Calf Intestinal Alkaline Phosphatase and their optimal reaction buffers to perform the dephosphorylation PROTOCOLS & APPLICATIONS GUIDE 6-11
Page 1 and 2:
CONTENTS I. Nucleic Acid Amplificat
Page 3 and 4:
| 1Nucleic Acid Amplification CONTE
Page 5 and 6:
| 1Nucleic Acid Amplification Unamp
Page 7 and 8:
| 1Nucleic Acid Amplification Capoz
Page 9 and 10:
| 1Nucleic Acid Amplification is co
Page 11 and 12:
| 1Nucleic Acid Amplification One i
Page 13 and 14:
| 1Nucleic Acid Amplification React
Page 15 and 16:
| 1Nucleic Acid Amplification comig
Page 17 and 18:
| 1Nucleic Acid Amplification inclu
Page 19 and 20:
| 1Nucleic Acid Amplification polym
Page 21 and 22:
| 1Nucleic Acid Amplification Addit
Page 23 and 24:
| 1Nucleic Acid Amplification 3. Pl
Page 25 and 26:
| 1Nucleic Acid Amplification 13. S
Page 27 and 28:
| 1Nucleic Acid Amplification IX. R
Page 29 and 30:
| 1Nucleic Acid Amplification Hoppe
Page 31 and 32:
|| 2RNA Interference CONTENTS I. RN
Page 33 and 34:
|| 2RNA Interference zebrafish (War
Page 35 and 36:
|| 2RNA Interference Additional Res
Page 37 and 38:
|| 2RNA Interference 2. Combine sep
Page 39 and 40:
|| 2RNA Interference Target Site Se
Page 41 and 42:
|| 2RNA Interference Transient Tran
Page 43 and 44:
|| 2RNA Interference VII. Reference
Page 45 and 46:
|| 2RNA Interference Wianny, F. and
Page 47 and 48:
||| 3Apoptosis I. Introduction A. C
Page 49 and 50:
||| 3Apoptosis ER stress pathway (H
Page 51 and 52:
||| 3Apoptosis pathways and demonst
Page 53 and 54:
||| 3Apoptosis Buffer Substrate Tha
Page 55 and 56:
||| 3Apoptosis System, Fluorometric
Page 57 and 58:
||| 3Apoptosis Qi, H. et al. (2003)
Page 59 and 60:
||| 3Apoptosis 2. Deparaffinize by
Page 61 and 62:
||| 3Apoptosis • 0.1% Triton® X-
Page 63 and 64:
||| 3Apoptosis 2. Pre-equilibrate t
Page 65 and 66:
||| 3Apoptosis Fluorescence (560/59
Page 67 and 68:
||| 3Apoptosis Ellis, H.M. and Horv
Page 69 and 70:
|||| 4Cell Viability CONTENTS I. In
Page 71 and 72:
|||| 4Cell Viability Table 4.1. Com
Page 73 and 74:
|||| 4Cell Viability E. Homogeneous
Page 75 and 76:
|||| 4Cell Viability equilibration
Page 77 and 78: |||| 4Cell Viability Microbial Grow
Page 79 and 80: |||| 4Cell Viability Materials Requ
Page 81 and 82: |||| 4Cell Viability Additional Res
Page 83 and 84: |||| 4Cell Viability GF-AFC substra
Page 85 and 86: |||| 4Cell Viability Fluorescence (
Page 87 and 88: |||| 4Cell Viability 5. Optional: I
Page 89 and 90: |||| 4Cell Viability Online Tools C
Page 91 and 92: |||| 4Cell Viability 5. Shake gentl
Page 93 and 94: |||| 4Cell Viability Crouch, S.P.M.
Page 95 and 96: ||||| 5Protein Expression I. Introd
Page 97 and 98: ||||| 5Protein Expression B. DNA-Dr
Page 99 and 100: ||||| 5Protein Expression Coupled S
Page 101 and 102: ||||| 5Protein Expression Standard
Page 103 and 104: ||||| 5Protein Expression mRNAs com
Page 105 and 106: ||||| 5Protein Expression Oxidative
Page 107 and 108: ||||| 5Protein Expression • radio
Page 109 and 110: ||||| 5Protein Expression Circular
Page 111 and 112: ||||| 5Protein Expression A. B. C.
Page 113 and 114: ||||| 5Protein Expression To reduce
Page 115 and 116: ||||| 5Protein Expression Figure 5.
Page 117 and 118: ||||| 5Protein Expression Snyder, M
Page 119 and 120: |||||| 6Expression Analysis I. Intr
Page 121 and 122: |||||| 6Expression Analysis synthes
Page 123 and 124: |||||| 6Expression Analysis Note: W
Page 125 and 126: |||||| 6Expression Analysis Note: N
Page 127: |||||| 6Expression Analysis require
Page 131 and 132: |||||| 6Expression Analysis PBS (1L
Page 133 and 134: ||||||| 7Cell Signaling CONTENTS I.
Page 135 and 136: ||||||| 7Cell Signaling SMALL GTP-B
Page 137 and 138: ||||||| 7Cell Signaling HO S N N S
Page 139 and 140: ||||||| 7Cell Signaling PN083 Intro
Page 141 and 142: ||||||| 7Cell Signaling III. Radioa
Page 143 and 144: ||||||| 7Cell Signaling CPM per Squ
Page 145 and 146: ||||||| 7Cell Signaling A. Nitrocel
Page 147 and 148: ||||||| 7Cell Signaling (continued
Page 149 and 150: ||||||| 7Cell Signaling Promega Pub
Page 151 and 152: ||||||| 7Cell Signaling Nonphosphor
Page 153 and 154: ||||||| 7Cell Signaling De Meyts, P
Page 155 and 156: |||||||| 8Bioluminescence Reporters
Page 167 and 168: Table 8.1. pGL4 Luciferase Reporter
Page 171 and 172: ||||||||| 9DNA Purification I. Intr
Page 173 and 174: ||||||||| 9DNA Purification Ideal f
Page 175 and 176: ||||||||| 9DNA Purification with a
Page 177 and 178: ||||||||| 9DNA Purification Culture
Page 179 and 180:
||||||||| 9DNA Purification C. Appr
Page 181 and 182:
||||||||| 9DNA Purification PureYie
Page 183 and 184:
||||||||| 9DNA Purification These a
Page 185 and 186:
||||||||| 9DNA Purification msp2 us
Page 187 and 188:
||||||||| 9DNA Purification Figure
Page 189 and 190:
||||||||| 9DNA Purification meaning
Page 191 and 192:
||||||||| 9DNA Purification D. Magn
Page 193 and 194:
||||||||| 9DNA Purification The Max
Page 195 and 196:
||||||||| 9DNA Purification Citatio
Page 197 and 198:
||||||||| 9DNA Purification VII. Ge
Page 199 and 200:
||||||||| 9DNA Purification 8. Cent
Page 201 and 202:
||||||||| 9DNA Purification Combine
Page 203 and 204:
||||||||| 9DNA Purification A. B. P
Page 205 and 206:
||||||||| 9DNA Purification 3. Appl
Page 207 and 208:
||||||||| 9DNA Purification van Sch
Page 209 and 210:
|||||||||| 10Cell Imaging I. Introd
Page 211 and 212:
|||||||||| 10Cell Imaging pFC14 CMV
Page 213 and 214:
|||||||||| 10Cell Imaging A. B. Fig
Page 215 and 216:
|||||||||| 10Cell Imaging 1. Cells
Page 217 and 218:
|||||||||| 10Cell Imaging technolog
Page 219 and 220:
|||||||||| 10Cell Imaging 8. Add ce
Page 221 and 222:
|||||||||| 10Cell Imaging 9. Drain
Page 223 and 224:
|||||||||| 10Cell Imaging and is me
Page 225 and 226:
|||||||||| 10Cell Imaging Anti-VACh
Page 227 and 228:
||||||||||| 11Protein Purification
Page 229 and 230:
Page 231 and 232:
Page 233 and 234:
Page 235 and 236:
Page 237 and 238:
Page 239 and 240:
Page 241 and 242:
Page 243 and 244:
Page 245 and 246:
Page 247 and 248:
Page 249 and 250:
Page 251 and 252:
Page 253 and 254:
Page 255 and 256:
Page 257 and 258:
|||||||||||| 12Transfection I. Intr
Page 259 and 260:
|||||||||||| 12Transfection Another
Page 261 and 262:
|||||||||||| 12Transfection C. Numb
Page 263 and 264:
|||||||||||| 12Transfection For a d
Page 265 and 266:
|||||||||||| 12Transfection • adh
Page 267 and 268:
|||||||||||| 12Transfection D. Endp
Page 269 and 270:
|||||||||||| 12Transfection 1X PBS
Page 271 and 272:
||||||||||||| 13Cloning CONTENTS I.
Page 273 and 274:
||||||||||||| 13Cloning allows a ra
Page 275 and 276:
||||||||||||| 13Cloning X-Gal (Cat.
Page 277 and 278:
||||||||||||| 13Cloning 1257bp PCR
Page 279 and 280:
||||||||||||| 13Cloning Figure 13.7
Page 281 and 282:
||||||||||||| 13Cloning Alkaline Ph
Page 283 and 284:
||||||||||||| 13Cloning Promega Pub
Page 285 and 286:
||||||||||||| 13Cloning Ligation 1.
Page 287 and 288:
||||||||||||| 13Cloning 7. Recommen
Page 289 and 290:
||||||||||||| 13Cloning Figure 13.9
Page 291 and 292:
||||||||||||| 13Cloning 1. Use the
Page 293 and 294:
||||||||||||| 13Cloning 4. Briefly
Page 295 and 296:
|||||||||||||| 14DNA-Based Human Id
Page 297 and 298:
Page 299 and 300:
Page 301 and 302:
Page 303 and 304:
Page 305 and 306:
Page 307 and 308:
show all

Protocols and Applications Guide (US Letter Size) - Promega

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?