ChIP/Liver harvest

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(Materials)
(Procedure)
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==Procedure==
==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==
==Notes==

Revision as of 14:17, 6 December 2006

Contents

Overview

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

Materials

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

  • PBS
  • PBS/1% Formaldehyde
  • Avertin (Anesthesia)

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)

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