Difference between revisions of "Chromatin Immunoprecipitation"

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(→‎Couple the primary antibody for each transcription factor or chromatin protein to magnetic beads)
 
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Line 22: Line 22:
 
* Dynabeads (Invitrogen cat#)
 
* Dynabeads (Invitrogen cat#)
 
* PBS with 5 mg/mL BSA and 1x Protease inhibitor (cold)
 
* PBS with 5 mg/mL BSA and 1x Protease inhibitor (cold)
−
* [LiCl Wash Buffer]] make fresh (cold)
+
* [[Dilution Buffer]]
−
* TE: 10 mM Tris 7.5, 0.1 mM EDTA (cold)
+
*[[Low Salt Immune Complex Wash Buffer]]
 +
* [[High Salt Immune Complex Wash Buffer]]
 +
* [[LiCl Immune Complex Wash Buffer]]  
 +
* [[TE Buffer]]
 
* [[ChIP Elution Buffer]] make fresh
 
* [[ChIP Elution Buffer]] make fresh
 +
* RNase A (10ug/ul;-20C)
 +
* Proteinase K (10ug/ul; -20C)
 
* QIAquick PCR Purification Kit
 
* QIAquick PCR Purification Kit
  
Line 64: Line 69:
  
 
11. Resuspend pellet to 1 ml with RIPA Buffer in a '''15 mL falcon tube''' (Do not vortex the tubes and try to avoid bubbles. Bubbles will cause popping and loss of samples during sonication).
 
11. Resuspend pellet to 1 ml with RIPA Buffer in a '''15 mL falcon tube''' (Do not vortex the tubes and try to avoid bubbles. Bubbles will cause popping and loss of samples during sonication).
 +
''Note: Remember to take a portion of this resuspended pellet prior to lysing to compare unsheared to sheared DNA.''
  
 
12. Using the Sonics VibraCell Sonicator, sonicate each 1.0 ml ChIP sample on ice, in a cold room, at Power Output 5 watts 6 times for 30 seconds each  
 
12. Using the Sonics VibraCell Sonicator, sonicate each 1.0 ml ChIP sample on ice, in a cold room, at Power Output 5 watts 6 times for 30 seconds each  
−
(60% amplitude), with at least 30 second cooling on ice between each 30-second sanitation. Remember to clean sonicator with water prior to use, in between samples and following use.
+
(~45-50% amplitude), with at least 30 second cooling on ice between each 30-second sanitation. Remember to clean sonicator with water prior to use, in between samples and following use.
 
*If using the Branson Sonifier 250: Set at constant cycle, output control 3 (will give output measurement of 5) and sonicate samples 10x each for 10 sec with a 20 sec recovery period between each.
 
*If using the Branson Sonifier 250: Set at constant cycle, output control 3 (will give output measurement of 5) and sonicate samples 10x each for 10 sec with a 20 sec recovery period between each.
  
Line 74: Line 80:
  
 
===Immunoprecipitation===
 
===Immunoprecipitation===
 +
(taken from Millipore EZ ChIP protocol)
  
 
''Perform all steps in an ice bucket or in the cold room at 4°C.''
 
''Perform all steps in an ice bucket or in the cold room at 4°C.''
  
 
==== Couple the primary antibody for each transcription factor or chromatin protein to magnetic beads====
 
==== Couple the primary antibody for each transcription factor or chromatin protein to magnetic beads====
−
# Add 20 μl re-suspended magnetic bead slurry (for each sample you plan to use with this antibody)  to a 1.5 ml microfuge tube on ice containing 1 ml cold PBS/BSA. Mix well, '''do not vortex'''. ''Note: For each wash, you will want to invert the tube while on the magnet rack to collect the beads that remain in the liquid at the top of the tube. This will allow you aspirate the liquid from the top of the tube between washes without sucking up any of the beads.''
+
15. Prepare enough Dilution Buffer containing protease inhibitors for the number of desired immunoprecipitations and store on ice.  
−
# Place the microfuge tubes on the magnet rack and remove supernatants.
+
−
# Resuspend the beads in 1 ml cold PBS/BSA.
+
−
# Repeat Steps b and c 3 times.
+
−
# Add 200 μl PBS/BSA (for each sample you plan to use this antibody with) to beads.
+
−
# Add 1 μg primary antibody. Mix gently by tapping--'''Do not vortex beads'''.
+
−
# Gently mix on a rotator platform for at least '''2 hours''' at 4°C.
+
−
# Wash beads 3 times (steps b-c), resuspending the beads by inverting the tubes during each wash.
+
−
# Resuspend in 100 μl  PBS/BSA (for each sample you plan to use this antibody with), and proceed to Step 2.
+
  
−
==== Incubate bead-antibody complex with fragmented, cross-linked chromatin====
+
16. Each IP requires the addition of 900 μL of Dilution Buffer and 4.5 μL of Protease Inhibitor Cocktail II.  
−
# Add 100 μl of antibody-coupled beads from above to each 25ug chromatin of preparation (from Sonication protocol) and incubate on a rotator for '''1 hour at room temperature''', followed by '''1 hour at  4°C'''.
+
−
# Collect beads containing immuno-bound chromatin by placing the microfuge tube on a magnet rack.
+
−
# Remove and discard supernatant. ''Turn water bath to 65°C before next step''.
+
−
# Wash beads 5 times with 1 mL LiCl Wash Buffer, mixing 3 minutes for each wash on a rotator.
+
−
# Add 1 ml TE Buffer to beads. Mix 1 minute on rotator and then place tubes on magnet rack to collect beads and discard supernatant.
+
−
# Resuspend the bead pellet in 200 μl IP Elution Buffer (at room temperature). Vortex to mix.
+
  
−
==== Reverse cross-linking and recover ChIP DNA ====
+
17. Immunoprecipitations should include the positive control (Anti-RNA Polymerase II), and the negative control, (Normal Mouse IgG), and the antibody of interest (user supplied). It is recommended that the user include a negative control IgG of the same species as the antibody of interest.
−
# Incubate beads from above in a 65°C water bath for '''1 hour''', shake or vortex every 15 minutes to elute the immuno-bound chromatin from the beads.
+
* Prepare one microfuge tube containing 100 μL of sheared crosslinked chromatin (Section B, step 5) for the number of desired immunoprecipitations and put on ice. If chromatin has been previously frozen, thaw on ice.
−
# Spin at 14,000 rpm in a microfuge at room temperature for 3 minutes.
+
* Alternatively, if multiple immunoprecipitations will be performed from the same chromatin preparation, place the entire volume for the number of desired immunoprecipitations in one large tube that will be able to accommodate a volume of 1.1 mL for each IP.
−
# Collect the supernatant, which contains the ChIP’d DNA. The tubes can be placed on the magnet to facilitate supernatant recovery.
+
* Each 100 μL will contain ~1 x 106 cell equivalents of chromatin.
−
# Incubate the supernatant containing the ChIP’d DNA in a 65°C water bath overnight to complete the reversal of the formaldehyde cross-links.
+
 
 +
18. Add 900 μL of Dilution Buffer containing Protease Inhibitor Cocktail II into each tube containing 100 μL of chromatin.
 +
* Alternatively, if multiple immunoprecipitations will be performed from the same chromatin preparation, use the appropriate volume of Dilution Buffer containing Protease Inhibitor Cocktail II for the correct number of immunoprecipitations.
 +
 
 +
19. Add 60 μL of Protein G Agarose for each IP.
 +
* The Protein G Agarose is a 50% slurry. Gently mix by inversion before pipetting.
 +
* This step serves to “preclear” the chromatin, i.e., to remove proteins or DNA that may bind nonspecifically to the Protein G agarose.
 +
* Alternatively, if multiple immunoprecipitations will be performed from the same chromatin preparation, use the appropriate volume of Protein G Agarose for the correct number of immunoprecipitations.
 +
 
 +
20. Incubate for '''1 hour''' at 4°C with rotation.
 +
 
 +
21. Pellet agarose by brief centrifugation (3000-5000 x g for 1 minute).
 +
 
 +
* Do not spin Protein G Agarose beads at high speeds. Applying excessive g-force may crush or deform the beads and cause them to pellet inconsistently.
 +
22. Remove 10 μL (1%) of the supernatant as Input and save at 4°C until Section D, step 1.
 +
* If different chromatin preparations are being carried together through this protocol, remove
 +
1% of the chromatin as Input from each.
 +
 
 +
23. Collect the remaining supernatant and dispense 1 mL aliquots into fresh microfuge tubes. Discard agarose pellet.
 +
 
 +
24. Add the immunoprecipitating antibody to the supernatant fraction:
 +
* For the positive control, anti-RNA Polymerase, add 1.0 μg of antibody per tube.
 +
* For the negative control, Normal Mouse IgG, add 1.0 μg of antibody per tube.
 +
* For user-provided antibody and controls, add between 1-10 μg of antibody per tube. The appropriate amount of antibody needs to be determined empirically.
 +
 
 +
25. Incubate '''overnight''' at 4°C with rotation.
 +
* It may be possible to reduce the incubation time of the IP. This depends on many factors
 +
(antibody, gene target, cell type, etc.) and will have to be tested empirically.
 +
 
 +
26. Add 60 μL of Protein G Agarose to each IP and incubate for '''1 hour''' at 4°C with rotation.
 +
* This serves to collect the antibody/antigen/DNA complex.
 +
 
 +
27. Pellet Protein G Agarose by brief centrifugation (3000-5000 x g for 1 minute) and remove the
 +
supernatant fraction.
 +
 
 +
28. Wash the Protein G Agarose-antibody/chromatin complex by resuspending the beads in 1 mL each of the cold buffers in the order listed below and incubating for '''3-5 minutes''' on a rotating platform followed by brief centrifugation (3000-5000 x g for 1 minute) and careful removal of the supernatant fraction:
 +
** [[Low Salt Immune Complex Wash Buffer]] (Catalog # 20-154), '''one wash'''
 +
** [[High Salt Immune Complex Wash Buffer]] (Catalog # 20-155), '''one wash'''
 +
** [[LiCl Immune Complex Wash Buffer]] (Catalog # 20-156), '''3-5 washes'''
 +
** [[TE Buffer]] (Catalog # 20-157), '''two washes''' ''Note: for TE washes use a pipette to carefully aspirate, the beads seem to come off of the magnet easily with this wash''
 +
 
 +
=== Elution of Protein/DNA Complexes ===
 +
===== Prior to starting this section: =====
 +
* Bring 1 M NaHCO3 to room temperature. A precipitate may be observed but will go into solution once room temperature is achieved. The 1 M NaHCO3 can be vortexed.
 +
* Set water bath to 65°C for use later.
 +
29. Make Elution Buffer for all IP tubes as well as all Input tubes.
 +
* For each tube, prepare 200 μL of elution buffer as follows: 10 μL 20% SDS, 20 μL 1 M NaHCO3 and 170 μL sterile, distilled water.
 +
* OR can make this way [[ChIP Elution Buffer]]
 +
* Alternatively, make a large volume to accommodate all tubes. For example, if there are 10 tubes mix together 105 μL 20% SDS, 210 μL 1M NaHCO3 and 1.785 mL sterile, distilled water.
 +
 
 +
30. For Input tubes, add 200 μL of Elution Buffer and set aside at room temperature.
 +
 
 +
31. Add 100 μL of Elution Buffer to each tube containing the antibody/agarose complex. Mix by flicking tube gently.
 +
 
 +
32. Incubate at room temperature for '''15 minutes'''.
 +
 
 +
33. Pellet agarose by brief centrifugation (3000-5000 x g for 1 minute) and collect supernatant into new microfuge tubes.
 +
 
 +
34. Repeat steps 4-6 and combine eluates (total volume = 200 μL).
 +
 
 +
=== Reverse Crosslinks of Protein/DNA Complexes to Free DNA===
 +
36. To all tubes (IPs and Inputs) add 8 μL 5 M NaCl and incubate at 65°C for 4-5 hours or '''overnight''' to reverse the DNA – Protein crosslinks. After this step the sample can be stored at -20°C and the protocol continued the next day.
 +
 
 +
37. To all tubes, add 1 μL of RNase A and incubate for '''30 minutes''' at 37°C.
 +
 
 +
38. Add 4 μL 0.5M EDTA, 8 μL 1M Tris-HCl and 1 μL Proteinase K to each tube and incubate at 45°C for
 +
'''1-2 hours'''.
  
 
==== Purification of ChIP DNA ====
 
==== Purification of ChIP DNA ====
−
# Add 5 volumes Qiagen Buffer PB (QIAquick PCR Purification Kit) to one volume of ChIP’d DNA. Add pH detector (at a 1:250 dilution) to samples. Upon addition of Buffer PB, the sample should be yellow, indicating the correct pH. If the sample is not yellow, the pH should be adjusted with 3M sodium acetate as recommended by the manufacturer (Qiagen). One microliter at a time, mixing between each works fine.
+
39. Add 5 volumes Qiagen Buffer PB (QIAquick PCR Purification Kit) to one volume of ChIP’d DNA. Add pH detector (at a 1:250 dilution) to samples. Upon addition of Buffer PB, the sample should be yellow, indicating the correct pH. If the sample is not yellow, the pH should be adjusted with 3M sodium acetate as recommended by the manufacturer (Qiagen). One microliter at a time, mixing between each works fine.
−
# Add half (~600 µl) of the solution to a QIAquick PCR Purification column, centrifuge for 30-60 sec @ 13,000 RPM , and then repeat with other half to bind the ~1.2 ml sample on a Qiagen column.
+
 
−
# Wash the column with 750 µl Qiagen Buffer PE, centrifuge for 30-60sec @ 13,000 RPM.
+
40. Add half (~600 µl) of the solution to a QIAquick PCR Purification column, centrifuge for 30-60 sec @ 13,000 RPM , and then repeat with other half to bind the ~1.2 ml sample on a Qiagen column.
−
# Empty the collection tube and centrifuge the column containing the bound DNA a second time to allow it to dry.
+
 
−
# Elute the DNA from the column with two 35 µl aliquots (note: this is how much you will need to run duplicates with 5 primers and may need to be adjusted based on your experiment) of warmed (~55°C) Qiagen Buffer EB, allow to sit on column for 1 minute, spin for 1 min @ 13,000 RPM, and repeat).
+
41. Wash the column with 750 µl Qiagen Buffer PE, centrifuge for 30-60sec @ 13,000 RPM.
 +
 
 +
42. Empty the collection tube and centrifuge the column containing the bound DNA a second time to allow it to dry.
 +
 
 +
43. Elute the DNA from the column with two 35 µl aliquots (note: this is how much you will need to run duplicates with 5 primers and may need to be adjusted based on your experiment) of warmed (~55°C) Qiagen Buffer EB, allow to sit on column for 1 minute, spin for 1 min @ 13,000 RPM, and repeat).
  
 
===Analysis of Immunoprecipitated DNA===
 
===Analysis of Immunoprecipitated DNA===
 
* See [[RT-PCR primer design for ChIP]] to design primers if analysing by qPCR
 
* See [[RT-PCR primer design for ChIP]] to design primers if analysing by qPCR

Latest revision as of 15:23, 10 May 2018


FOR CELL CULTURE SAMPLES

This protocol is modified from the Myer's Lab ChIPseq protocol v011014 found here. The original citation for this methodology is:

Johnson DS, Mortazavi A, Myers RM, Wold B. Genome-Wide Mapping of in Vivo Protein-DNA Interactions. Science (80- ) 316: 1497–1502, 2007. doi:10.1126/science.1141319

Before You Start

Buffers and Solutions Needed

Equipment

  • Cool microfuge and swinging bucket centrifuge down to 4C

Protocol

This protocol involves preparation of the crosslinked DNA, immunoprecipitation of the DNA and analysis by qPCR. It is possible to stop and freeze the samples after each of these steps.

Crosslinking, Lysis and Shearing of DNA

1. Remove culture plates from the incubator and place at room temperature on the bench.

2. Add formaldehyde to a final concentration of 1% directly to the media of adherent cells growing on tissue culture plates, swirl gently, and incubate at room temperature for 10 minutes.

  • If using 10cm dishes add 250ul of 40% formaldehyde

3. Stop the cross-linking reaction by adding glycine to a final concentration of 0.125M and swirl gently to mix.

  • If using 10cm dishes add 0.5mL of the 2.5M glycine stock solution

4. Remove media from plates and wash cells with equal volume cold (4°C) 1X PBS.

  • 10mL for 10cm dish

5. Aspirate the PBS and add 2.5 ml cold (4°C) Farnham lysis buffer (make sure to add PI).

6. Scrape the cells off the plate with a cell scraper and transfer into 15-ml conical tubes on ice.

7. Pellet cells at 2,000 rpm for 5 minutes at 4°C.

8. Place cells on ice. Carefully remove supernatant and either proceed to sonication step or snap-freeze in liquid nitrogen and store at -80°C or in liquid nitrogen.

9. Resuspend each fresh or frozen pellet (containing 2 x 107 cells) on ice in 1 ml Farnham Lysis Buffer and mix gently by flicking the test tube. Briefly homogenize cells by running the cells through a 18-gauge needle ~10X. Note: This treatment breaks the cells while keeping the nuclei mostly intact.

10. Collect the crude nuclear prep by centrifuging at 2,000 rpm at 4°C for 5 minutes.

11. Resuspend pellet to 1 ml with RIPA Buffer in a 15 mL falcon tube (Do not vortex the tubes and try to avoid bubbles. Bubbles will cause popping and loss of samples during sonication). Note: Remember to take a portion of this resuspended pellet prior to lysing to compare unsheared to sheared DNA.

12. Using the Sonics VibraCell Sonicator, sonicate each 1.0 ml ChIP sample on ice, in a cold room, at Power Output 5 watts 6 times for 30 seconds each (~45-50% amplitude), with at least 30 second cooling on ice between each 30-second sanitation. Remember to clean sonicator with water prior to use, in between samples and following use.

  • If using the Branson Sonifier 250: Set at constant cycle, output control 3 (will give output measurement of 5) and sonicate samples 10x each for 10 sec with a 20 sec recovery period between each.

13. Spin the sonicated mixture at 14,000 rpm in a microfuge for 15 minutes at 4°C and collect the supernatant and nano drop samples and calculate the amount needed for 25ug of chromatin.

14. Snap-freeze the sample in liquid nitrogen and store at -80°C, or do not freeze and continue with the immunoprecipitation steps below.

Immunoprecipitation

(taken from Millipore EZ ChIP protocol)

Perform all steps in an ice bucket or in the cold room at 4°C.

Couple the primary antibody for each transcription factor or chromatin protein to magnetic beads

15. Prepare enough Dilution Buffer containing protease inhibitors for the number of desired immunoprecipitations and store on ice.

16. Each IP requires the addition of 900 μL of Dilution Buffer and 4.5 μL of Protease Inhibitor Cocktail II.

17. Immunoprecipitations should include the positive control (Anti-RNA Polymerase II), and the negative control, (Normal Mouse IgG), and the antibody of interest (user supplied). It is recommended that the user include a negative control IgG of the same species as the antibody of interest.

  • Prepare one microfuge tube containing 100 μL of sheared crosslinked chromatin (Section B, step 5) for the number of desired immunoprecipitations and put on ice. If chromatin has been previously frozen, thaw on ice.
  • Alternatively, if multiple immunoprecipitations will be performed from the same chromatin preparation, place the entire volume for the number of desired immunoprecipitations in one large tube that will be able to accommodate a volume of 1.1 mL for each IP.
  • Each 100 μL will contain ~1 x 106 cell equivalents of chromatin.

18. Add 900 μL of Dilution Buffer containing Protease Inhibitor Cocktail II into each tube containing 100 μL of chromatin.

  • Alternatively, if multiple immunoprecipitations will be performed from the same chromatin preparation, use the appropriate volume of Dilution Buffer containing Protease Inhibitor Cocktail II for the correct number of immunoprecipitations.

19. Add 60 μL of Protein G Agarose for each IP.

  • The Protein G Agarose is a 50% slurry. Gently mix by inversion before pipetting.
  • This step serves to “preclear” the chromatin, i.e., to remove proteins or DNA that may bind nonspecifically to the Protein G agarose.
  • Alternatively, if multiple immunoprecipitations will be performed from the same chromatin preparation, use the appropriate volume of Protein G Agarose for the correct number of immunoprecipitations.

20. Incubate for 1 hour at 4°C with rotation.

21. Pellet agarose by brief centrifugation (3000-5000 x g for 1 minute).

  • Do not spin Protein G Agarose beads at high speeds. Applying excessive g-force may crush or deform the beads and cause them to pellet inconsistently.

22. Remove 10 μL (1%) of the supernatant as Input and save at 4°C until Section D, step 1.

  • If different chromatin preparations are being carried together through this protocol, remove

1% of the chromatin as Input from each.

23. Collect the remaining supernatant and dispense 1 mL aliquots into fresh microfuge tubes. Discard agarose pellet.

24. Add the immunoprecipitating antibody to the supernatant fraction:

  • For the positive control, anti-RNA Polymerase, add 1.0 μg of antibody per tube.
  • For the negative control, Normal Mouse IgG, add 1.0 μg of antibody per tube.
  • For user-provided antibody and controls, add between 1-10 μg of antibody per tube. The appropriate amount of antibody needs to be determined empirically.

25. Incubate overnight at 4°C with rotation.

  • It may be possible to reduce the incubation time of the IP. This depends on many factors

(antibody, gene target, cell type, etc.) and will have to be tested empirically.

26. Add 60 μL of Protein G Agarose to each IP and incubate for 1 hour at 4°C with rotation.

  • This serves to collect the antibody/antigen/DNA complex.

27. Pellet Protein G Agarose by brief centrifugation (3000-5000 x g for 1 minute) and remove the supernatant fraction.

28. Wash the Protein G Agarose-antibody/chromatin complex by resuspending the beads in 1 mL each of the cold buffers in the order listed below and incubating for 3-5 minutes on a rotating platform followed by brief centrifugation (3000-5000 x g for 1 minute) and careful removal of the supernatant fraction:

Elution of Protein/DNA Complexes

Prior to starting this section:
  • Bring 1 M NaHCO3 to room temperature. A precipitate may be observed but will go into solution once room temperature is achieved. The 1 M NaHCO3 can be vortexed.
  • Set water bath to 65°C for use later.

29. Make Elution Buffer for all IP tubes as well as all Input tubes.

  • For each tube, prepare 200 μL of elution buffer as follows: 10 μL 20% SDS, 20 μL 1 M NaHCO3 and 170 μL sterile, distilled water.
  • OR can make this way ChIP Elution Buffer
  • Alternatively, make a large volume to accommodate all tubes. For example, if there are 10 tubes mix together 105 μL 20% SDS, 210 μL 1M NaHCO3 and 1.785 mL sterile, distilled water.

30. For Input tubes, add 200 μL of Elution Buffer and set aside at room temperature.

31. Add 100 μL of Elution Buffer to each tube containing the antibody/agarose complex. Mix by flicking tube gently.

32. Incubate at room temperature for 15 minutes.

33. Pellet agarose by brief centrifugation (3000-5000 x g for 1 minute) and collect supernatant into new microfuge tubes.

34. Repeat steps 4-6 and combine eluates (total volume = 200 μL).

Reverse Crosslinks of Protein/DNA Complexes to Free DNA

36. To all tubes (IPs and Inputs) add 8 μL 5 M NaCl and incubate at 65°C for 4-5 hours or overnight to reverse the DNA – Protein crosslinks. After this step the sample can be stored at -20°C and the protocol continued the next day.

37. To all tubes, add 1 μL of RNase A and incubate for 30 minutes at 37°C.

38. Add 4 μL 0.5M EDTA, 8 μL 1M Tris-HCl and 1 μL Proteinase K to each tube and incubate at 45°C for 1-2 hours.

Purification of ChIP DNA

39. Add 5 volumes Qiagen Buffer PB (QIAquick PCR Purification Kit) to one volume of ChIP’d DNA. Add pH detector (at a 1:250 dilution) to samples. Upon addition of Buffer PB, the sample should be yellow, indicating the correct pH. If the sample is not yellow, the pH should be adjusted with 3M sodium acetate as recommended by the manufacturer (Qiagen). One microliter at a time, mixing between each works fine.

40. Add half (~600 µl) of the solution to a QIAquick PCR Purification column, centrifuge for 30-60 sec @ 13,000 RPM , and then repeat with other half to bind the ~1.2 ml sample on a Qiagen column.

41. Wash the column with 750 µl Qiagen Buffer PE, centrifuge for 30-60sec @ 13,000 RPM.

42. Empty the collection tube and centrifuge the column containing the bound DNA a second time to allow it to dry.

43. Elute the DNA from the column with two 35 µl aliquots (note: this is how much you will need to run duplicates with 5 primers and may need to be adjusted based on your experiment) of warmed (~55°C) Qiagen Buffer EB, allow to sit on column for 1 minute, spin for 1 min @ 13,000 RPM, and repeat).

Analysis of Immunoprecipitated DNA

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