Protocols and Applications Guide (US Letter Size) - Promega

More documents

Recommendations

Info

||||||||||||| 13Cloning Figure 13.5. Features of the Flexi® Vectors. Figure 13.6. PCR primer design. The PmeI site appends a single valine codon at the 3´ end of the protein-coding region and allows either termination or readthrough to append a carboxy-terminal peptide, depending on the vector backbone. Protocols & Applications Guide www.promega.com rev. 3/09 PROTOCOLS & APPLICATIONS GUIDE 13-7
||||||||||||| 13Cloning Figure 13.7. Cloning a protein-coding region into the Flexi® Vectors. PCR primers are designed to append SgfI and PmeI sites onto the protein-coding region. After amplification, the PCR product is purified to remove the DNA polymerase and primers and digested with SgfI and PmeI. The DNA is purified again to remove the small oligonucleotides released by the restriction enzymes. The digested PCR product is ligated into an acceptor Flexi® Vector that has been digested with SgfI and PmeI. Following transformation, the cells are selected with the appropriate antibiotic for the particular Flexi® Vector used. Unlike site-specific recombination vector systems, the Flexi® Vector Systems do not require appending multiple amino acids to the amino or carboxy termini of the protein of interest (Figure 13.6). In addition, the systems do not require an archival entry vector, and most applications allow direct entry into the vector suited to the experimental design (e.g., mammalian expression or N-terminal, glutathione-S-transferase (GST) fusion vectors). For instance, you might clone your PCR product into the pFN2A (GST) Flexi® Vector to express your GST-tagged protein in E. coli for purification. However, an easy transfer of your insert after SgfI/PmeI digest followed by ligation into the pF4K CMV Flexi® Vector will allow you to transfect the same protein-coding region into a mammalian cell and determine its expression level. Protocols & Applications Guide www.promega.com rev. 3/09 Any Flexi® Vector can act as an acceptor of a protein-coding region flanked by SgfI and PmeI sites (Figure 13.7). The SgfI site is upstream of the start codon of the protein-coding region, and depending upon the Flexi® Vector used for cloning, this allows the expression of a native (untagged) protein or an amino (N)-terminal-tagged protein by readthrough of the SgfI site. The PmeI site contains the stop codon for the protein-coding region and appends a single valine residue to the carboxy (C)-terminus of the protein (Figure 13.6). The C-terminal Flexi® Vectors allow expression of C-terminal-tagged proteins. While these vectors can act as acceptors of protein-coding regions flanked by SgfI and PmeI, they lack a PmeI site and contain a different blunt-ended site, EcoICRI. Inserts cloned using these sites cannot be removed from the C-terminal Flexi® Vectors and transferred to other Flexi® Vectors (Figure 13.4, Panel B). Additional Resources for the Flexi® Vector Systems Technical Bulletins and Manuals TM254 Flexi® Vector Systems Technical Manual (www.promega.com /tbs/tm254/tm254.html) Promega Publications CN011 Clone and express protein-coding regions using the Flexi® Vector Systems (www.promega.com /cnotes/cn011/cn011_13.htm) PN094 The next-generation assay for mammalian protein interactions: The CheckMate/Flexi® Vector Mammalian Two-Hybrid System (www.promega.com /pnotes/94/14410_12/14410_12.html) PN093 The Flexi® Vector Systems: The easy way to clone (www.promega.com /pnotes/93/14023_08/14023_08.html) PN091 Metal affinity tag for protein expression and purification using the Flexi® Vectors (www.promega.com /pnotes/91/12972_17/12972_17.html) PN089 A new system for cloning and expressing protein-coding regions (www.promega.com /pnotes/89/12416_11/12416_11.html) Online Tools Flexi® Vector Systems Animation (www.promega.com /paguide/animation/selector.htm?coreName=flexi01) Flexi® Vector Primer Design Tool (www.promega.com /techserv/tools/flexivector/) PROTOCOLS & APPLICATIONS GUIDE 13-8
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 and 128:
|||||| 6Expression Analysis require
Page 129 and 130:
|||||| 6Expression Analysis For a p
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 157 and 158:
Page 159 and 160:
Page 161 and 162:
Page 163 and 164:
Page 165 and 166:
Page 167 and 168:
Table 8.1. pGL4 Luciferase Reporter
Page 169 and 170:
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 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: ||||||||||||| 13Cloning 1257bp PCR
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
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?