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'''<span style="font-size:18.0pt;line-height:107%">Protocol for Genomic Library Screening </span>'''
=1.  PCR DIG Labeling =
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).
''' '''
This is the standard method, for alternative labeling method, please see #2.
(For reagent and more details, please refer to kit protocol at –
[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.
Steps:
<span style="line-height:111%">1)<span style="font:7.0pt "Times New Roman"">    </span></span>Thaw the reagents and store on ice;
Briefly vortex and centrifuge all reagents before setting up the reactions.
<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
<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
{|
!width="20%"| '''<span style="font-size:11.0pt;line-height:107%"><span style="mso-spacerun:yes">Reagent </span></span>'''
!width="20%"| '''<span style="font-size:11.0pt;line-height:107%">Volume Required for DIG-labeled experimental probe </span>'''
!width="20%"| '''<span style="font-size:11.0pt;line-height:107%">Volumne Required for Unlabeled control probe </span>'''
!width="20%"| '''<span style="font-size:11.0pt;line-height:
  107%">Volume Required for DIG labeled <span class="SpellE">tPA</span> control probe </span>'''
!width="20%"| '''<span style="font-size:11.0pt;line-height:107%"><span style="mso-spacerun:yes">Final concentration </span></span>'''
|-
| <span style="font-size:11.0pt;line-height:
  107%">Water PCR-grade </span>
| <span style="font-size:
  11.0pt;line-height:107%">Add up to 50ul </span>
| <span style="font-size:11.0pt;line-height:
  107%">Add up to 50ul </span>
| <span style="font-size:11.0pt;line-height:
  107%">Add up to 50ul </span>
| <span style="font-size:11.0pt;line-height:
  107%"><span style="mso-spacerun:yes"> </span></span>
|-
|
<span style="font-size:11.0pt;line-height:
  107%">PCR buffer w/ </span>
<span style="font-size:11.0pt;line-height:
  107%">MgCl2 (10X) </span>
<span style="font-size:11.0pt;line-height:
  107%">(vial3) </span>
| <span style="font-size:
  11.0pt;line-height:107%">5ul </span>
| <span style="font-size:11.0pt;line-height:
  107%">5ul </span>
| <span style="font-size:11.0pt;line-height:
  107%">5ul </span>
| <span style="font-size:11.0pt;line-height:
  107%">1X </span>
|-
| <span style="font-size:11.0pt;line-height:
  107%">PCR DIG probe synthesis Mix (vial2) </span>
| <span style="font-size:
  11.0pt;line-height:107%">5ul </span>
| <span style="font-size:11.0pt;line-height:
  107%">- </span>
| <span style="font-size:11.0pt;line-height:
  107%">5ul </span>
|
<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>
<span style="font-size:11.0pt;line-height:
  107%">DIG-<span class="SpellE">dUTP</span> </span>
|-
|
<span class="SpellE"><span style="font-size:
  11.0pt;line-height:107%">dNTP</span></span><span style="font-size:11.0pt;
  line-height:107%"> stock </span>
<span style="font-size:11.0pt;line-height:
  107%">solution (vial4) </span>
| <span style="font-size:
  11.0pt;line-height:107%">- </span>
| <span style="font-size:11.0pt;line-height:
  107%">5ul </span>
| <span style="font-size:11.0pt;line-height:
  107%">- </span>
| <span style="font-size:11.0pt;line-height:
  107%">200uM each <span class="SpellE">dNTP</span> </span>
|-
| <span style="font-size:11.0pt;line-height:
  107%">Forward PCR </span>
| <span style="font-size:
  11.0pt;line-height:107%">5ul </span>
| <span style="font-size:11.0pt;line-height:
  107%">5ul </span>
| <span style="font-size:11.0pt;line-height:
  107%">5ul </span>
| <span style="font-size:11.0pt;line-height:
  107%">0.1-1uM </span>
|-
| <span style="font-size:11.0pt;line-height:
  107%">primer 10X conc. </span>
|
|
|
|
|-
| <span style="font-size:11.0pt;line-height:
  107%">Reverse PCR primer 10X </span>
| <span style="font-size:
  11.0pt;line-height:107%">5ul </span>
| <span style="font-size:11.0pt;line-height:
  107%">5ul </span>
| <span style="font-size:11.0pt;line-height:
  107%"><span style="mso-spacerun:yes"> </span></span>
| <span style="font-size:11.0pt;line-height:
  107%">0.1-1uM </span>
|-
|
<span style="font-size:11.0pt;line-height:
  107%">Enzyme mix </span>
<span style="font-size:11.0pt;line-height:
  107%">(vial1) </span>
| <span style="font-size:
  11.0pt;line-height:107%">0.75ul </span>
| <span style="font-size:11.0pt;line-height:
  107%">0.75ul </span>
| <span style="font-size:11.0pt;line-height:
  107%">0.75ul </span>
| <span style="font-size:11.0pt;line-height:
  107%"><span style="mso-spacerun:yes"> </span></span>
|-
| <span style="font-size:11.0pt;line-height:
  107%">template </span>
| <span style="font-size:
  11.0pt;line-height:107%">variable </span>
| <span style="font-size:11.0pt;line-height:
  107%">variable </span>
| <span style="font-size:11.0pt;line-height:
  107%">5ul </span>
|
<span style="font-size:11.0pt;line-height:
  107%">1-50ng genomic DNA/ </span>
<span style="font-size:11.0pt;line-height:107%">10-100pg plasmid DNA </span>
|-
| '''<span style="font-size:11.0pt;line-height:107%">Total Volume </span>'''
| <span style="font-size:
  11.0pt;line-height:107%">50ul </span>
| <span style="font-size:11.0pt;line-height:
  107%">50ul </span>
| <span style="font-size:11.0pt;line-height:
  107%">50ul </span>
| <span style="font-size:11.0pt;line-height:
  107%"><span style="mso-spacerun:yes"> </span></span>
|}
<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.
Note—Adjusting the DIG-dUTP concentration:
<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.
<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).
<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.
Ping’s note--
<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 .
<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))
<span style="line-height:111%">5)<span style="font:7.0pt "Times New Roman"">    </span></span>PCR program--
95°C 5min
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'''''
<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'''''
72°C 5min
8°C  Forever
<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.
<span style="line-height:111%">7)<span style="font:7.0pt "Times New Roman"">    </span></span>Gel check
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.
<span style="font-size:11.0pt;line-height:107%;font-family:
"Calibri",sans-serif">[[Image:Digscreen_image1.JPG]]</span>
= 2. DIG high prime DNA label =
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
Digoxigenin-11-dUTP, labeling grade Klenow enzyme and an optimized reaction buffer.
(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.
<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.
<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.
'''''Note''''': Complete denaturation is essential for efficient labeling.
<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.
'''''Note:''''' Longer incubations (up to 20 h) will increase the yield of DIG-labeled DNA.
<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.
'''''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.
''' '''
= 3.<span style="font-weight:normal"> </span>Determination of labeling efficiency-Dilution and dot blot =
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.
(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.
<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.
<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.
<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.
<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'''.
<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.'''
<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.
<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'''.
<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.
'''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.
<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'':'''
Drying of the membrane during exposure will result in dark background.
<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.
'''''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.
<span style="font-size:11.0pt;line-height:107%;font-family:
"Calibri",sans-serif">[[Image:DIGscreen image2.jpg|410x262px]]</span>
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. ''''''
= 4. Screen library =
''' '''
(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.
1) Stripping filters
'''''(Important Tip: Membranes should never be allowed to dry before stripping.''''' ''Once dried, the membrane cannot be stripped and reprobed.)''
Stripping DIG-labeled DNA Probe after Chemiluminescent Detection
<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
<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.
<span style="line-height:111%">•<span style="font:7.0pt "Times New Roman"">      </span></span>Rinse membrane with 2× SSC for 5 min
<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
<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
<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
mix by inversion (avoid foaming) to form the hybridization solution
<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
<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
<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
<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
= 5. Immunological Detection =
(Please refer to protocol of ''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.
<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'''.
<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
<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.
<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.'''
<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.
<span style="line-height:111%">16)<span style="font:7.0pt "Times New Roman"">  </span></span>Expose 30min & develop film.
[[Image:DIGscreen image3.jpg]]<span style="font-size:11.0pt;line-height:
107%"> </span>
'''6. Check the correct probe for the positive clone''' 1) Pick up all positive clones and mini prep.
2) Put positive clones DNA  to make dots in small filter
Redo the hybrization (steps 4(2) until the end) with individual probes to tell which probe this clone is responding to.

Revision as of 13:49, 11 May 2020