Drosophila CHiP on chip: Difference between revisions

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'''Important''':  This protocol is dynamic, feel free to discuss any stages and make changes as you see fit.  If you use are currently using this protocol in the lab then it is important that you make a note of the date of the version that you are using and return to this if you need to repeat the experiment.  You can find the history by clicking on the history tab above.  The history never changes, allowing you  reference a particular protocol at a snap shot in time.
'''Important''':  This protocol is dynamic, feel free to discuss any stages and make changes as you see fit.  If you use are currently using this protocol in the lab then it is important that you make a note of the date of the version that you are using and return to this if you need to repeat the experiment.  You can find the history by clicking on the history tab above.  The history never changes, allowing you  reference a particular protocol at a snap shot in time.


==Protocols==
==Protocols== from: Version 1.1. R. Auburn. (15−03−2006)
# Incubate embryos at 37 °C for 15 minutes to heat shock the embryos (omit this step if non−heat shocked embryos are required). #Dechorionate embryos for 3 minutes in a solution of weak bleach (5% w/w available chlorine) at
#Incubate embryos at 37 °C for 15 minutes to heat shock the embryos (omit this step if non−heat shocked embryos are required).  
room temperature.  
#Dechorionate embryos for 3 minutes in a solution of weak bleach (5% w/w available chlorine) at room temperature.  
# Wash well with tap water and place onto filter paper. Transfer to fresh filter paper to weigh. Transfer 1−2 g of embryos to a 50 ml Falcon tube and add 50 ml PBS/Triton to wash the embryos.
#Wash well with tap water and place onto filter paper.  
Centrifuge for 1 minute at 500 x g (1680 rpm) to pellet embryos.
#Transfer to fresh filter paper to weigh.  
#Transfer 1−2 g of embryos to a 50 ml Falcon tube and add 50 ml PBS/Triton to wash the embryos.  
#Centrifuge for 1 minute at 500 x g (1680 rpm) to pellet embryos.
#Discard supernatant and add 10 ml cross−linking solution, 487 μl 40% formaldehyde and 30 ml n−heptane. Shake vigorously (by hand works well...but tiring) at room temperature for 15 minutes.
#Discard supernatant and add 10 ml cross−linking solution, 487 μl 40% formaldehyde and 30 ml n−heptane. Shake vigorously (by hand works well...but tiring) at room temperature for 15 minutes.
# Centrifuge for 1 minute at 500 x g (1680 rpm) to pellet embryos and discard supernatant.
#Centrifuge for 1 minute at 500 x g (1680 rpm) to pellet embryos and discard supernatant.
Resuspend in 30 ml PBS/Glycine/Triton, best to resuspend in 3 ml of buffer and then add the rest. Then allow the embryos to sediment.
#Resuspend in 30 ml PBS/Glycine/Triton, best to resuspend in 3 ml of buffer and then add the rest. Then allow the embryos to sediment.
# Remove supernatant and add 50 ml ice−cold PBS/Triton and resuspend. Best to add a small volume of the buffer to resuspend the embryos. Allow the embryos to sediment.
#Remove supernatant and add 50 ml ice−cold PBS/Triton and resuspend. Best to add a small volume of the buffer to resuspend the embryos. Allow the embryos to sediment.
# Remove supernatant and resuspend in 15 ml ice−cold PBS/Triton + protease inhibitors, again resuspend in a small volume and then add remainder. Dounce with a Wheaton homogeniser pestle B.
#Remove supernatant and resuspend in 15 ml ice−cold PBS/Triton + protease inhibitors, again resuspend in a small volume and then add remainder. Dounce with a Wheaton homogeniser pestle B.
# Centrifuge at 400 x g (1500 rpm) for 1 minute and transfer supernatant to a fresh tube. Centrifuge at 1100 x g (2495 rpm) for 10 minutes at 4 °C and discard supernatant. Resuspend in 15ml ice−cold Cell Lysis Buffer with protease inhibitors, best to resuspend in a small volume and then add remainder. Dounce with a Wheaton homogeniser pestle A. Transfer 2 equal aliquots into 13.5 ml
#Centrifuge at 400 x g (1500 rpm) for 1 minute and transfer supernatant to a fresh tube. Centrifuge at 1100 x g (2495 rpm) for 10 minutes at 4 °C and discard supernatant. Resuspend in 15ml ice−cold Cell Lysis Buffer with protease inhibitors, best to resuspend in a small volume and then add remainder. Dounce with a Wheaton homogeniser pestle A. Transfer 2 equal aliquots into 13.5 ml screw−capped conical base tubes.
screw−capped conical base tubes.
#Centrifuge at 2000 x g (3365 rpm) for 4 minutes at 4 °C to pellet the nuclei. Resuspend in 1 ml of ice−cold Nuclear Lysis Buffer with protease inhibitors, incubate for 20 minutes at 4 °C.
# Centrifuge at 2000 x g (3365 rpm) for 4 minutes at 4 °C to pellet the nuclei. Resuspend in 1 ml of ice−cold Nuclear Lysis Buffer with protease inhibitors, incubate for 20 minutes at 4 °C.
#Add 2 ml ice−cold Nuclear Lysis Buffer with protease inhibitors and 0.3 g acid washed 212−300 micron glass beads.
# Add 2 ml ice−cold Nuclear Lysis Buffer with protease inhibitors and 0.3 g acid washed 212−300 micron glass beads.
#Sonicate on ice following the regime below to produce chromatin fragments with an average size of 500 bp using a sonicator fitted with a microtip. This step should be calibrated for individual sonicators to generate chromatin of an appropriate size.
# Sonicate on ice following the regime below to produce chromatin fragments with an average size of 500 bp using a sonicator fitted with a microtip. This step should be calibrated for individual sonicators to generate chromatin of an appropriate size.
#*1 x 30 seconds on level 3
#*1 x 30 seconds on level 3
#*5 x 30 seconds on level 4
#*5 x 30 seconds on level 4

Revision as of 07:46, 18 April 2006

If you are interested in doing CHiP chip with Drosophila then please feel free to contribute to the protocols found here. The original protocol was added and adapted with permission from Rob White's lab. You can download the original from www.FlyChip.org.uk

Important: This protocol is dynamic, feel free to discuss any stages and make changes as you see fit. If you use are currently using this protocol in the lab then it is important that you make a note of the date of the version that you are using and return to this if you need to repeat the experiment. You can find the history by clicking on the history tab above. The history never changes, allowing you reference a particular protocol at a snap shot in time.

==Protocols== from: Version 1.1. R. Auburn. (15−03−2006)

  1. Incubate embryos at 37 °C for 15 minutes to heat shock the embryos (omit this step if non−heat shocked embryos are required).
  2. Dechorionate embryos for 3 minutes in a solution of weak bleach (5% w/w available chlorine) at room temperature.
  3. Wash well with tap water and place onto filter paper.
  4. Transfer to fresh filter paper to weigh.
  5. Transfer 1−2 g of embryos to a 50 ml Falcon tube and add 50 ml PBS/Triton to wash the embryos.
  6. Centrifuge for 1 minute at 500 x g (1680 rpm) to pellet embryos.
  7. Discard supernatant and add 10 ml cross−linking solution, 487 μl 40% formaldehyde and 30 ml n−heptane. Shake vigorously (by hand works well...but tiring) at room temperature for 15 minutes.
  8. Centrifuge for 1 minute at 500 x g (1680 rpm) to pellet embryos and discard supernatant.
  9. Resuspend in 30 ml PBS/Glycine/Triton, best to resuspend in 3 ml of buffer and then add the rest. Then allow the embryos to sediment.
  10. Remove supernatant and add 50 ml ice−cold PBS/Triton and resuspend. Best to add a small volume of the buffer to resuspend the embryos. Allow the embryos to sediment.
  11. Remove supernatant and resuspend in 15 ml ice−cold PBS/Triton + protease inhibitors, again resuspend in a small volume and then add remainder. Dounce with a Wheaton homogeniser pestle B.
  12. Centrifuge at 400 x g (1500 rpm) for 1 minute and transfer supernatant to a fresh tube. Centrifuge at 1100 x g (2495 rpm) for 10 minutes at 4 °C and discard supernatant. Resuspend in 15ml ice−cold Cell Lysis Buffer with protease inhibitors, best to resuspend in a small volume and then add remainder. Dounce with a Wheaton homogeniser pestle A. Transfer 2 equal aliquots into 13.5 ml screw−capped conical base tubes.
  13. Centrifuge at 2000 x g (3365 rpm) for 4 minutes at 4 °C to pellet the nuclei. Resuspend in 1 ml of ice−cold Nuclear Lysis Buffer with protease inhibitors, incubate for 20 minutes at 4 °C.
  14. Add 2 ml ice−cold Nuclear Lysis Buffer with protease inhibitors and 0.3 g acid washed 212−300 micron glass beads.
  15. Sonicate on ice following the regime below to produce chromatin fragments with an average size of 500 bp using a sonicator fitted with a microtip. This step should be calibrated for individual sonicators to generate chromatin of an appropriate size.
    • 1 x 30 seconds on level 3
    • 5 x 30 seconds on level 4
    • Between each sonication rest on ice for 90 seconds
  16. Transfer to microfuge tubes and centrifuge at 16,000 x g (13000 rpm) in a microfuge for 10 minutes at 4 °C.
  17. Take the supernatant (fixed sheared chromatin) and transfer to cryotubes, e.g., 200 μl aliquots, and flash freeze in liquid nitrogen. Store at −80 °C.

News

  1. The protocol is not complete, I'm currently moving it from the PDF to the web, I'll be adding details for collection of chromatin from whole flies rather than from embryos (the original protocol).

Questions

  1. please add Q & A here

Recent changes/changes from original flychip.org

  1. please add any changes here

Contacts

  1. --Johncumbers 10:31, 18 April 2006 (EDT)
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