imported>Echinobase |
imported>Ctelmer |
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| '''<span style="font-size:18.0pt;line-height:107%">Protocol for Genomic Library Screening </span>'''
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| = 1. PCR DIG Labeling =
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| The DIG Nonradioactive System from Roche is the basis for our screening protocol. It is a sensitive method for nucleic acid labeling and detection. The labeled probe can be used to detect target nucleic acid (RNA or DNA) on a membrane (Southern, Northern, or dot blot).
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| ''' '''
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| This is the standard method, for alternative labeling method, please see #2.
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| (For reagent and more details, please refer to kit protocol at –
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| [http://www.roche-applied-science.com/PROD_INF/MANUALS/DIG_MAN/dig_toc.htm <span style="color:purple">http://www.roche-applied-science.com/PROD_INF/MANUALS/DIG_MAN/dig_toc.htm</span>][http://www.roche-applied-science.com/PROD_INF/MANUALS/DIG_MAN/dig_toc.htm <span style="color:black;text-decoration:none">)</span>] For each experimental or control sample, add the following components to a sterile microcentrifuge tube. Place the tube on ice during pipetting.
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| Steps:
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| <span style="line-height:111%">1)<span style="font:7.0pt "Times New Roman""> </span></span>Thaw the reagents and store on ice;
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| Briefly vortex and centrifuge all reagents before setting up the reactions.
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| <span style="line-height:149%">2)<span style="font:7.0pt "Times New Roman""> </span></span>Prepare a 10X concentration solution (1-10ul) of the forward and reverse PCR primer
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| <span style="line-height:111%">3)<span style="font:7.0pt "Times New Roman""> </span></span>For each 50ul reaction, add the components in the order listed below to a sterile reaction tube on ice
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| {|
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| !width="20%"| '''<span style="font-size:11.0pt;line-height:107%"><span style="mso-spacerun:yes">Reagent </span></span>'''
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| !width="20%"| '''<span style="font-size:11.0pt;line-height:107%">Volume Required for DIG-labeled experimental probe </span>'''
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| !width="20%"| '''<span style="font-size:11.0pt;line-height:107%">Volumne Required for Unlabeled control probe </span>'''
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| !width="20%"| '''<span style="font-size:11.0pt;line-height:
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| 107%">Volume Required for DIG labeled <span class="SpellE">tPA</span> control probe </span>'''
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| !width="20%"| '''<span style="font-size:11.0pt;line-height:107%"><span style="mso-spacerun:yes">Final concentration </span></span>'''
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| |-
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| | <span style="font-size:11.0pt;line-height:
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| 107%">Water PCR-grade </span>
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| | <span style="font-size:
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| 11.0pt;line-height:107%">Add up to 50ul </span>
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| | <span style="font-size:11.0pt;line-height:
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| 107%">Add up to 50ul </span>
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| | <span style="font-size:11.0pt;line-height:
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| 107%">Add up to 50ul </span>
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| | <span style="font-size:11.0pt;line-height:
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| 107%"><span style="mso-spacerun:yes"> </span></span>
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| |-
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| <span style="font-size:11.0pt;line-height:
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| 107%">PCR buffer w/ </span>
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| <span style="font-size:11.0pt;line-height:
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| 107%">MgCl2 (10X) </span>
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| <span style="font-size:11.0pt;line-height:
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| 107%">(vial3) </span>
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| | <span style="font-size:
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| 11.0pt;line-height:107%">5ul </span>
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| | <span style="font-size:11.0pt;line-height:
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| 107%">5ul </span>
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| | <span style="font-size:11.0pt;line-height:
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| 107%">5ul </span>
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| | <span style="font-size:11.0pt;line-height:
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| 107%">1X </span>
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| |-
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| | <span style="font-size:11.0pt;line-height:
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| 107%">PCR DIG probe synthesis Mix (vial2) </span>
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| | <span style="font-size:
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| 11.0pt;line-height:107%">5ul </span>
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| | <span style="font-size:11.0pt;line-height:
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| 107%">- </span>
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| | <span style="font-size:11.0pt;line-height:
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| 107%">5ul </span>
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| <span style="font-size:11.0pt;line-height:99%">200uM <span class="SpellE">dATP,dCTP,dGTP</span>, 130uM dTTP,70 <span class="SpellE">uM</span> </span>
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| <span style="font-size:11.0pt;line-height:
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| 107%">DIG-<span class="SpellE">dUTP</span> </span>
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| |-
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| <span class="SpellE"><span style="font-size:
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| 11.0pt;line-height:107%">dNTP</span></span><span style="font-size:11.0pt;
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| line-height:107%"> stock </span>
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| <span style="font-size:11.0pt;line-height:
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| 107%">solution (vial4) </span>
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| | <span style="font-size:
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| 11.0pt;line-height:107%">- </span>
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| | <span style="font-size:11.0pt;line-height:
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| 107%">5ul </span>
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| | <span style="font-size:11.0pt;line-height:
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| 107%">- </span>
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| | <span style="font-size:11.0pt;line-height:
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| 107%">200uM each <span class="SpellE">dNTP</span> </span>
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| |-
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| | <span style="font-size:11.0pt;line-height:
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| 107%">Forward PCR </span>
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| | <span style="font-size:
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| 11.0pt;line-height:107%">5ul </span>
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| | <span style="font-size:11.0pt;line-height:
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| 107%">5ul </span>
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| | <span style="font-size:11.0pt;line-height:
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| 107%">5ul </span>
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| | <span style="font-size:11.0pt;line-height:
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| 107%">0.1-1uM </span>
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| |-
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| | <span style="font-size:11.0pt;line-height:
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| 107%">primer 10X conc. </span>
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| |-
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| | <span style="font-size:11.0pt;line-height:
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| 107%">Reverse PCR primer 10X </span>
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| | <span style="font-size:
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| 11.0pt;line-height:107%">5ul </span>
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| | <span style="font-size:11.0pt;line-height:
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| 107%">5ul </span>
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| | <span style="font-size:11.0pt;line-height:
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| 107%"><span style="mso-spacerun:yes"> </span></span>
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| | <span style="font-size:11.0pt;line-height:
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| 107%">0.1-1uM </span>
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| |-
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| <span style="font-size:11.0pt;line-height:
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| 107%">Enzyme mix </span>
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| <span style="font-size:11.0pt;line-height:
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| 107%">(vial1) </span>
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| | <span style="font-size:
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| 11.0pt;line-height:107%">0.75ul </span>
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| | <span style="font-size:11.0pt;line-height:
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| 107%">0.75ul </span>
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| | <span style="font-size:11.0pt;line-height:
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| 107%">0.75ul </span>
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| | <span style="font-size:11.0pt;line-height:
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| 107%"><span style="mso-spacerun:yes"> </span></span>
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| |-
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| | <span style="font-size:11.0pt;line-height:
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| 107%">template </span>
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| | <span style="font-size:
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| 11.0pt;line-height:107%">variable </span>
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| | <span style="font-size:11.0pt;line-height:
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| 107%">variable </span>
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| | <span style="font-size:11.0pt;line-height:
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| 107%">5ul </span>
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| <span style="font-size:11.0pt;line-height:
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| 107%">1-50ng genomic DNA/ </span>
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| <span style="font-size:11.0pt;line-height:107%">10-100pg plasmid DNA </span>
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| | '''<span style="font-size:11.0pt;line-height:107%">Total Volume </span>'''
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| | <span style="font-size:
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| 11.0pt;line-height:107%">50ul </span>
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| | <span style="font-size:11.0pt;line-height:
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| 107%">50ul </span>
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| | <span style="font-size:11.0pt;line-height:
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| 107%">50ul </span>
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| | <span style="font-size:11.0pt;line-height:
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| 107%"><span style="mso-spacerun:yes"> </span></span>
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| |}
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| <span style="line-height:150%">4)<span style="font:7.0pt "Times New Roman""> </span></span>Mix the reagents and centrifuge briefly to collect the sample at the bottom of the tube.
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| Note—Adjusting the DIG-dUTP concentration:
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| <span style="line-height:149%">•<span style="font:7.0pt "Times New Roman""> </span></span>The final concentration of DIG-dUTP (70uM), when using the undiluted PCR DIC probe Synthesis Mix (vial2), works well for labeling probes up to 1kb long.
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| <span style="line-height:149%">•<span style="font:7.0pt "Times New Roman""> </span></span>For labeling probes 1-3kb long, reduce the final concentration of DIG-dUTP to 35uM; Mix equal parts of PCR DIG probe synthesis Mix (vial 2) and dNTP stock solution (vial 4).
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| <span style="line-height:149%">•<span style="font:7.0pt "Times New Roman""> </span></span>For labeling short probes (<1Kb) of high GC content it might also be necessary to reduce the DIG-dUTP concentration to 35uM.
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| Ping’s note--
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| <span style="line-height:149%">•<span style="font:7.0pt "Times New Roman""> </span></span>For template, use water diluted cDNA mini prep product (25pg/ul). For above 50ml reaction, use 1 ul .
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| <span style="line-height:111%">•<span style="font:7.0pt "Times New Roman""> </span></span>Use (35uM DIG-dUTP) (2.5 ul of DIG probe mix (vial 2) + 2.5ul of dNTP (vial 4))
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| <span style="line-height:111%">5)<span style="font:7.0pt "Times New Roman""> </span></span>PCR program--
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| 95°C 5min
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| 95°C 30s<span style="font-family:Wingdings">à</span>55°C 30s <span style="font-family:Wingdings">à</span>72°C (time base on product) '''''10 cycles'''''
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| <span style="font-size:11.0pt;line-height:111%;font-family:"Calibri",sans-serif"> </span>95°C 30s<span style="font-family:Wingdings">à</span>55°C 30s<span style="font-family:Wingdings">à</span>72°C (time base on product) + 5s/cycle '''''20 cycles'''''
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| 72°C 5min
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| 8°C Forever
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| <span style="line-height:111%">6)<span style="font:7.0pt "Times New Roman""> </span></span>For PCR product, clean up with <span style="color:red">zoymo DNA clean</span>. Check OD260.
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| <span style="line-height:111%">7)<span style="font:7.0pt "Times New Roman""> </span></span>Gel check
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| Run 2ul of labeled and unlabeled PCR onto 1% agarose gel to check the label (see picture below). If label is OK, use 40ul/20ml of labeled probe for screening.
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| <span style="font-size:11.0pt;line-height:107%;font-family:
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| "Calibri",sans-serif">[[Image:Digscreen_image1.JPG]]</span>
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| '''2. DIG high prime DNA label''':
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| DNA is random primed labeled with Digoxigenin-11-dUTP using DIG-High Prime, a 5 × concentrated labeling mixture of random hexamers, dNTP mix containing alkali-labile
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| Digoxigenin-11-dUTP, labeling grade Klenow enzyme and an optimized reaction buffer.
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| (For reagent and more details, please refer to ''DIG-High Prime DNA Labeling and Detection Starter'' Kit I at [http://www.genomex.com/Downloads/Sequencing_Site.pdf <span style="color:blue">http://www.genomex.com/Downloads/Sequencing_Site.pdf</span>][http://www.genomex.com/Downloads/Sequencing_Site.pdf <span style="color:black;text-decoration:none">)</span>] *All solution comes from the Kit.
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| <span style="line-height:178%">1)<span style="font:7.0pt "Times New Roman""> </span></span>Add 1 μg '''template DNA''' (linear or supercoiled) and autoclaved, double distilled water to a final volume of 16 μl to a reaction vial.
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| <span style="line-height:149%">2)<span style="font:7.0pt "Times New Roman""> </span></span>Denature the DNA by heating in a boiling water bath for 10 min and quickly chilling in an ice/water bath.
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| '''''Note''''': Complete denaturation is essential for efficient labeling.
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| <span style="line-height:152%">3)<span style="font:7.0pt "Times New Roman""> </span></span>Mix '''DIG-High Prime''' (vial 1) thoroughly and add 4ul to the denatured DNA, mix and centrifuge briefly. Incubate for 1 h or O/N at 37°C.
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| '''''Note:''''' Longer incubations (up to 20 h) will increase the yield of DIG-labeled DNA.
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| <span style="line-height:154%">4)<span style="font:7.0pt "Times New Roman""> </span></span>Stop the reaction by adding 2ul 0.2 M '''EDTA''' (pH 8.0) and/or by heating to 65° C for 10 min.
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| '''''Note:''''' The lengths of the DIG labeled fragments obtained with DIG-High Prime range from 200 bp to 1000 bp or larger, depending on the lengths of the original template.
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| ''' '''
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| = 3.<span style="font-weight:normal"> </span>Determination of labeling efficiency-Dilution and dot blot =
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| Determination of the yield of DIG-labeled DNA is most important for optimal and reproducible hybridization results. Too high of a probe concentration in the hybridization mix causes background, while too low of a concentration leads to weak signals.
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| (For reagent and more details, please refer to ''DIG-High Prime DNA Labeling and Detection Starter'' Kit I at [http://www.genomex.com/Downloads/Sequencing_Site.pdf <span style="color:blue">http://www.genomex.com/Downloads/Sequencing_Site.pdf</span>][http://www.genomex.com/Downloads/Sequencing_Site.pdf <span style="color:black;text-decoration:none">)</span>] *All solution comes from the Kit.
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| <span style="line-height:152%">1)<span style="font:7.0pt "Times New Roman""> </span></span>Apply a 1μl spot of tubes 2-9 from your labeled probes and the labeled control to the nylon membrane.
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| <span style="line-height:149%">2)<span style="font:7.0pt "Times New Roman""> </span></span>Fix the nucleic acid to the membrane by cross linking with UV-light or baking for 30 min at 120°C.
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| <span style="line-height:152%">3)<span style="font:7.0pt "Times New Roman""> </span></span>Transfer the membrane into a plastic container with 20 ml '''Maleic acid buffer'''. Incubate under shaking for 2 min at 15-25°C.
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| <span style="line-height:111%">4)<span style="font:7.0pt "Times New Roman""> </span></span>Incubate for 30 min in 10 ml '''Blocking solution'''.
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| <span style="line-height:111%">5)<span style="font:7.0pt "Times New Roman""> </span></span>Incubate for 30 min in 10 ml '''Antibody solution.'''
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| <span style="line-height:111%">6)<span style="font:7.0pt "Times New Roman""> </span></span>Wash with 10 ml '''Washing buffer''', 2 × 15 min.
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| <span style="line-height:111%">7)<span style="font:7.0pt "Times New Roman""> </span></span>Equilibrate 2-5 min in 10 ml '''Detection buffer'''.
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| <span style="line-height:151%">8)<span style="font:7.0pt "Times New Roman""> </span></span>Place membrane with DNA side facing up on a development folder (or hybridization bag) and apply 0.1 ml '''CSPD ready-to-use''' (i.e. 4 drops from the dropper bottle 5) to the membrane.
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| '''Immediately''' cover the membrane with the second sheet of the folder to spread the substrate evenly and without air bubbles over the membrane. Incubate for 5 min at 15-25°C.
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| <span style="line-height:111%">9)<span style="font:7.0pt "Times New Roman""> </span></span>Squeeze out excess liquid and seal the edges of the development folder. '''''Note'':'''
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| Drying of the membrane during exposure will result in dark background.
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| <span style="line-height:149%">10)<span style="font:7.0pt "Times New Roman""> </span></span>Expose to an appropriate imager for 5-20 min or to X-ray film for 15-25 min at 1525°C.
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| '''''Note''''': Luminescence continues for at least 48 hours. The signal increases in the first few hours after initiation of the detection reaction until it will reach a plateau where signal intensity remains almost constant during the next 24 – 48 hours. Multiple exposures can be taken to achieve the desired signal strength.
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| <span style="font-size:11.0pt;line-height:107%;font-family:
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| "Calibri",sans-serif">[[Image:DIGscreen image2.jpg|410x262px]]</span>
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| Compare the intensity of the spots out of your labeling reaction to the control and calculate the amount of DIG-labeled DNA. If the 0.1 pg dilution spots of your probe and of the control are visible, then the labeled probe has reached the expected labeling efficiency. ''''''
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|
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| = <span style="font-weight:normal">4. </span>Screen library =
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| ''' '''
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| (For reagent and more details, please refer to ''DIG-High Prime DNA Labeling and Detection Starter'' Kit I at [http://www.genomex.com/Downloads/Sequencing_Site.pdf <span style="color:blue">http://www.genomex.com/Downloads/Sequencing_Site.pdf</span>][http://www.genomex.com/Downloads/Sequencing_Site.pdf <span style="color:black;text-decoration:none">)</span>] *All solution comes from the Kit.
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| 1) Stripping filters
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| '''''(Important Tip: Membranes should never be allowed to dry before stripping.''''' ''Once dried, the membrane cannot be stripped and reprobed.)''
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| Stripping DIG-labeled DNA Probe after Chemiluminescent Detection
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| <span style="line-height:111%">•<span style="font:7.0pt "Times New Roman""> </span></span>Rinse membrane thoroughly with bouble distilled water for 1 min
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| <span style="line-height:150%">•<span style="font:7.0pt "Times New Roman""> </span></span>Wash membrane twice at 37°C in Stripping Buffer (0.2 M NaOH containing 0.1% SDS) for 15 min each.
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| <span style="line-height:111%">•<span style="font:7.0pt "Times New Roman""> </span></span>Rinse membrane with 2× SSC for 5 min
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| <span style="line-height:111%">2)<span style="font:7.0pt "Times New Roman""> </span></span>Prehybrization in 40ml DIG Easy Hyb at 48°C for 1 hour
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| <span style="line-height:149%">3)<span style="font:7.0pt "Times New Roman""> </span></span>Denature the probe--all probes mixed in 350ul 10uM Tris and denatured under 95°C for 5min, then chill quickly on ice bath
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| <span style="line-height:149%">4)<span style="font:7.0pt "Times New Roman""> </span></span>Hybrization solution-- Immediately add the denatured probe to a tube containing the appropriate amount of prewarmed DIG Easy Hyb (3.5 ml per 100 cm2) and
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| mix by inversion (avoid foaming) to form the hybridization solution
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| <span style="line-height:154%">5)<span style="font:7.0pt "Times New Roman""> </span></span>Pour off the prehybridization buffer.<span style="color:#F46E20"> </span>'''Immediately''' add hybridization solution containing DIG-labeled probe and hybrization at 48°C for 6-16 hours
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| <span style="line-height:150%">6)<span style="font:7.0pt "Times New Roman""> </span></span>Wash with low Stringency Buffer (1L 2XSSC containing 0.1% SDS) at room temperature for 15min twice
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| <span style="line-height:111%">7)<span style="font:7.0pt "Times New Roman""> </span></span>Preheat high Stringency Buffer (1L 0.5XSSC containing 0.1% SDS) into 65°C
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| <span style="line-height:149%">8)<span style="font:7.0pt "Times New Roman""> </span></span>Wash with preheated high Stringency Buffer (1L 0.5XSSC containing 0.1% SDS) at 65°C for 15min twice
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| = 5. Immunological Detection =
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|
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| (Please refer to protocol of ''DIG-High Prime DNA Labeling and Detection Starter'' Kit I at
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|
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| [http://www.genomex.com/Downloads/Sequencing_Site.pdf <span style="color:blue">http://www.genomex.com/Downloads/Sequencing_Site.pdf</span>][http://www.genomex.com/Downloads/Sequencing_Site.pdf <span style="color:black;text-decoration:none">)</span>] *All solution comes from the Kit.
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| <span style="line-height:149%">9)<span style="font:7.0pt "Times New Roman""> </span></span>After hybridization and stringency washes, rinse membrane for 2 min in '''Washing buffer'''.
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| <span style="line-height:111%">10)<span style="font:7.0pt "Times New Roman""> </span></span>Incubate in 250 ml 1X'''Blocking solution''' for 1 hour under room temperature
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| <span style="line-height:153%">11)<span style="font:7.0pt "Times New Roman""> </span></span>Prepare '''Antibody solution''': Spin AP antibody at 10rpm for 5min and then add AP antibody into 100ml 1Xblocking buffer into 1:10000.
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| <span style="line-height:151%">12)<span style="font:7.0pt "Times New Roman""> </span></span>Incubate in 100ml '''Antibody solution''' for 1 hour 13) Wash in 500ml '''Washing buffer''' (2X SSC)15min twice 14) Equilibrate 5 min in 20ml '''Detection buffer.'''
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| <span style="line-height:153%">15)<span style="font:7.0pt "Times New Roman""> </span></span>Incubate membrane in 10 ml freshly prepared '''color substrate solution''' in a appropriate container in the dark for 5 min. '''Do not shake''' during color development.
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| <span style="line-height:111%">16)<span style="font:7.0pt "Times New Roman""> </span></span>Expose 30min & develop film.
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| [[Image:DIGscreen image3.jpg]]<span style="font-size:11.0pt;line-height:
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| 107%"> </span>
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| '''6. Check the correct probe for the positive clone''' 1) Pick up all positive clones and mini prep.
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| 2) Put positive clones DNA to make dots in small filter
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| Redo the hybrization (steps 4(2) until the end) with individual probes to tell which probe this clone is responding to.
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