ChIP/Liver harvest: Difference between revisions
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==Overview== | ==Overview== | ||
This is a protocol for harvesting mouse liver tissue for [[ChIP|chromatin immunoprecipitation]]. | This is a protocol for harvesting mouse liver tissue for [[ChIP|chromatin immunoprecipitation]]. <br> | ||
<br> | |||
''This protocol is incomplete. More to come. ~ [[User:Cconboy|cmc]] 14:19, 6 December 2006 (EST)'' | |||
== | ==Reagents== | ||
'''Buffer A (100mL total)'''<br> | |||
* 91mL of H2O<br> | |||
* 1.5mL of 1M Hepes<br> | |||
* 2mL of 3M KCl<br> | |||
* 300µL of 5M NaCl<br> | |||
* 40µL of 0.5M EDTA<br> | |||
* 100µL of 0.5M EGTA<br> | |||
* 16.2µL of 2.1M Sucrose<br> | |||
* 7.5µL of 2M BME<br> | |||
*5mL of 2.5M Glycine (to make 2.5M stock- 187.5 g/L)<br> | |||
**A stock of Liver Buffer A can be made without BME or Glycine and stored at room temp. Add BME and Glycine before use. | |||
<br> | |||
'''Buffer B (100mL total)'''<br> | |||
* 1.5mL of 1M Hepes<br> | |||
* 2mL of 3M KCl<br> | |||
* 300µm of 5M NaCl<br> | |||
* 20µm of 0.5M EDTA<br> | |||
* 20µm of 0.5M EGTA<br> | |||
* 100mL of 2.1M Sucrose<br> | |||
<br> | |||
'''Other''' | |||
* Avertin (Anesthesia) | |||
* PBS<br> | |||
* PBS/1% Formaldehyde<br> | |||
* 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== | ==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== |
Latest revision as of 12:36, 6 December 2006
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)