ChIP/Liver harvest

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==Overview==
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This is a protocol for harvesting mouse liver tissue for [[ChIP|chromatin immunoprecipitation]]. <br>
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<br>
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''This protocol is incomplete. More to come. ~ [[User:Cconboy|cmc]] 14:19, 6 December 2006 (EST)''
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==Reagents==
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'''Buffer A (100mL total)'''<br>
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* 91mL of H2O<br>
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* 1.5mL of 1M Hepes<br>
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* 2mL of 3M KCl<br>
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* 300µL of 5M NaCl<br>
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* 40µL of 0.5M EDTA<br>
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* 100µL of 0.5M EGTA<br>
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* 16.2µL of 2.1M Sucrose<br>
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* 7.5µL of 2M BME<br>
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*5mL of 2.5M Glycine (to make 2.5M stock- 187.5 g/L)<br>
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**A stock of Liver Buffer A can be made without BME or Glycine and stored at room temp. Add BME and Glycine before use.
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<br>
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'''Buffer B (100mL total)'''<br>
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* 1.5mL of 1M Hepes<br>
 +
* 2mL of 3M KCl<br>
 +
* 300µm of 5M NaCl<br>
 +
* 20µm of 0.5M EDTA<br>
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* 20µm of 0.5M EGTA<br>
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* 100mL of 2.1M Sucrose<br>
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<br>
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'''Other'''
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* Avertin (Anesthesia)
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* PBS<br>
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* PBS/1% Formaldehyde<br>
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* 2.5M Glycine
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==Supplies==
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Surgical Instruments
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* Scissors
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* Tweezers
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* Bulldog clamp
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* String
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Other:
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* 50mL conical tubes (1 per Liver)
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* 2 of 10cc syringe
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* 1 of 3cc syringe
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* 26g needle
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* Angiocatheter
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* Ice with bucket
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'''Interested in posting a protocol on OpenWetWare?  Here is a template to help you do so.'''   
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==Procedure==
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===Harvest===
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Total Time= ~20min/liver
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* Open anaesthetized animal posterior to anterior
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* Tie a loose knot of string around the superior vena cava in between the diaphragm and top of liver
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* Tie a loose knot of string around the inferior vena cava
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* Insert catheter into inferior vena cava from posterior to anterior and secure with bulldog clamp making sure that the end of the catheter is anterior to the kidney junction
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* Screw 10cc syringe with PBS onto catheter
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* Tighten both knots and cut the portal vein
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* Perfuse with PBS ''very''  slowly. (3-5 minutes)
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* Switch syringes and ''slowly''  perfuse with 10cc PBS/formaldehyde
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**The total time for both syringes of perfusion should be 5-10 minutes
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* Cut diaphragm and remove liver
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* Put liver into 50mL conical tube cap and dice into little pieces
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* Incubate liver for 10 minutes and then add to 6mL glycine solution on ice
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'''Click the view source tab and copy everything below this line.  Paste it into your new protocol page.  Then replace the text in this page with your own protocol.  Feel free to add or delete sections as appropriate.'''
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Notes:
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<!-- COPY EVERYHING BELOW HERE TO START YOUR OWN PROTOCOL!  -->
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* Keep Buffer A on ice and PBS and PBS/formaldehyde in 30o water bath
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* Do not use mice older than 3 months or if they are sick
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* Harvest even numbers of animals- 2, 4, 6
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==Overview==
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===Hepatocyte Purification===                             
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* Pour minced liver into dousing apparatus and rinse beaker with Buffer A and add to dounce
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* Dounce until liquefied
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* Filter through plastic filter and wash with Buffer A
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* Gently layer onto a 1.5mL cushion of 1:1 Buffer A and B in a Corex tube
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* Centrifuge in fixed-angle rotor at 10,000rpm for 10min at 40
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* Decant and discard supernatant
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* Gently resuspend pellet in 5-10mL PBS
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* Layer onto a 1.5mL cushion of 1:1 Buffer A and B in a Corex tube
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* Centrifuge at 5,000rpm for 10min at 40
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* Gently decant and discard supernatant
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* Resuspend pellet in 3mL of sonication buffer or resuspend pellet in 5mL of PBS or TBS, transfer to 15mL conical tube, centrifuge, decant supernatant and freeze pellet in  -800
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Replace this sentence with a brief description of the protocol and its goal.
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Notes:
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*Purification protocol should be done in the cold room on ice
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==Materials==
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List reagents, supplies and equipment necessary to perform the protocol here.  For those materials which have their own OWW pages, link to that page.  Alternatively, links to the suppliers' page on that material are also appropriate.
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*supply 1 (i.e. tubes of a certain size? spreaders?)
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*reagent 1
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*X &mu;L reagent 2
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**component A (reagent 2 is made up of multiple components)
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**component B
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*equipment 1
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*equipment 2
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==Procedure==
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#Step 1
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#Step 2
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#*Step 2 has some additional information that goes with it.  i.e. Keep at 4&deg;C.
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#Step 3
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##Step 3 has multiple sub-steps within it.
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##Enumerate each of those.
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==Notes==
==Notes==
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#List troubleshooting tips here. 
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* ''Please contribute user notes!''
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#You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
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#Anecdotal observations that might be of use to others can also be posted here. 
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Please sign your name to your note by adding <font face="courier"><nowiki>('''~~~~''')</nowiki></font> to the end of your tip.
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==References==
==References==
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'''Relevant papers and books'''
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* (Young Lab)
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<!-- If this protocol has papers or books associated with it, list those references here.  See the [[OpenWetWare:Biblio]] page for more information. -->
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<biblio>
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#Goldbeter-PNAS-1981 pmid=6947258
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#Jacob-JMB-1961 pmid=13718526
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#Ptashne-Genetic-Switch isbn=0879697164
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</biblio>
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==Contact==
==Contact==
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*Who has experience with this protocol?
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* [[User:Cconboy|cmc]]

Current revision

Contents

Overview

This is a protocol for harvesting mouse liver tissue for chromatin immunoprecipitation.

This protocol is incomplete. More to come. ~ cmc 14:19, 6 December 2006 (EST)

Reagents

Buffer A (100mL total)

  • 91mL of H2O
  • 1.5mL of 1M Hepes
  • 2mL of 3M KCl
  • 300µL of 5M NaCl
  • 40µL of 0.5M EDTA
  • 100µL of 0.5M EGTA
  • 16.2µL of 2.1M Sucrose
  • 7.5µL of 2M BME
  • 5mL of 2.5M Glycine (to make 2.5M stock- 187.5 g/L)
    • A stock of Liver Buffer A can be made without BME or Glycine and stored at room temp. Add BME and Glycine before use.


Buffer B (100mL total)

  • 1.5mL of 1M Hepes
  • 2mL of 3M KCl
  • 300µm of 5M NaCl
  • 20µm of 0.5M EDTA
  • 20µm of 0.5M EGTA
  • 100mL of 2.1M Sucrose


Other

  • Avertin (Anesthesia)
  • PBS
  • PBS/1% Formaldehyde
  • 2.5M Glycine

Supplies

Surgical Instruments

  • Scissors
  • Tweezers
  • Bulldog clamp
  • String

Other:

  • 50mL conical tubes (1 per Liver)
  • 2 of 10cc syringe
  • 1 of 3cc syringe
  • 26g needle
  • Angiocatheter
  • Ice with bucket

Procedure

Harvest

Total Time= ~20min/liver

  • Open anaesthetized animal posterior to anterior
  • Tie a loose knot of string around the superior vena cava in between the diaphragm and top of liver
  • Tie a loose knot of string around the inferior vena cava
  • Insert catheter into inferior vena cava from posterior to anterior and secure with bulldog clamp making sure that the end of the catheter is anterior to the kidney junction
  • Screw 10cc syringe with PBS onto catheter
  • Tighten both knots and cut the portal vein
  • Perfuse with PBS very slowly. (3-5 minutes)
  • Switch syringes and slowly perfuse with 10cc PBS/formaldehyde
    • The total time for both syringes of perfusion should be 5-10 minutes
  • Cut diaphragm and remove liver
  • Put liver into 50mL conical tube cap and dice into little pieces
  • Incubate liver for 10 minutes and then add to 6mL glycine solution on ice

Notes:

  • Keep Buffer A on ice and PBS and PBS/formaldehyde in 30o water bath
  • Do not use mice older than 3 months or if they are sick
  • Harvest even numbers of animals- 2, 4, 6

Hepatocyte Purification

  • Pour minced liver into dousing apparatus and rinse beaker with Buffer A and add to dounce
  • Dounce until liquefied
  • Filter through plastic filter and wash with Buffer A
  • Gently layer onto a 1.5mL cushion of 1:1 Buffer A and B in a Corex tube
  • Centrifuge in fixed-angle rotor at 10,000rpm for 10min at 40
  • Decant and discard supernatant
  • Gently resuspend pellet in 5-10mL PBS
  • Layer onto a 1.5mL cushion of 1:1 Buffer A and B in a Corex tube
  • Centrifuge at 5,000rpm for 10min at 40
  • Gently decant and discard supernatant
  • Resuspend pellet in 3mL of sonication buffer or resuspend pellet in 5mL of PBS or TBS, transfer to 15mL conical tube, centrifuge, decant supernatant and freeze pellet in -800

Notes:

  • Purification protocol should be done in the cold room on ice

Notes

  • Please contribute user notes!

References

  • (Young Lab)

Contact

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