ChIP protocol实验室成熟的CHIP-seq及CHIP-qPCR操作流程

ChIP protocol

Fixation type: Type I:

1% formaldehyde /PBS(100ml): 36.5%formaldehyde (F8775-500ml) 2.5ml diluted in 1XPBS 88.75 ml Room temperature, 15min Type II:

2-5mM DSG (stock 50mM: 10mg in 540ul of dry DMSO), Room temperature, 45min 1% formaldehyde /PBS, Room temperature, 15min Type III:

1% Glutaraldehyde solution

(Grade I, 25% in H2O, specially purified for use as an electron microscopy fixative) sigma G5882-100ml, Room temperature, 15min

Stock buffer: 20%SDS; 0.5M, pH8.0 EDTA; 1M, pH7.8 Tris-Cl; 10% TritonX100; 5M NaCl, Buffer recipe: Lysis Buffer 20%SDS 0.5M, pH8.0 EDTA 1M, pH7.8 Tris-Cl 50Xcocktail(Roche) Double Distilled water

Dilution Buffer 10% TritonX100 0.5M, pH8.0 EDTA 5M NaCl 1M, pH7.8 Tris-Cl 50Xcocktail(Roche) Double Distilled water 100ml 20%SDS 10% TritonX100 0.5M, pH8.0 EDTA 5M NaCl 1M, pH7.8 Tris-Cl 50Xcocktail(Roche) Double Distilled water TSE1 0.5ml 10ml 0.4ml 3ml 2ml 1ml(add freshly) 83.1ml TSE2 0.5ml 10ml 0.4ml 8ml 2ml 1ml(add freshly) 78.1ml

10ml 0.5ml 0.2ml 0.5ml 0.2ml(add freshly) 8.6ml 2ml 100ul 50ul 100ul 40ul(add freshly) 1720ul 500ml 50ml 2ml 15ml 10ml 10ml(add freshly) 413ml 100ml 10ml 0.4ml 3ml 2ml 2ml(add freshly) 82.6ml ChIP protocol

1.25M Glycine(MW: 75.07): 18.77g plus 200ml ddw 5M NaCl(MW58.45): 292.2g plus 1L ddw, autoclave 20% SDS(MW288.38): 20g plus 100ml ddw 1M, pH7.8 Tris-Cl: 121.1g plus 1L ddw, autoclave Option:

Drug treatment：10-7 M E2 treat 1hr

Day1: cell fixation, cell lysis, sonication and antibody incubation Collecting cells

1. After fixation, cold PBS wash twice

2. 1ml PBS to scrape cells and 0.5ml PBS scrape cells

3. Transfer cells into 1.5ml tubes, max speed 30sec~1min, centrifugation at 4℃

4. If you want to stop at step3, you can freeze the samples by liquid nitrogen quickly, then store at -80℃

for long time storage. If not, continue to step 5

Cell lysis and sonication

5. Add 300ul lysis buffer plus complete cocktail (avoid creating bubbles)

6. Sonication（声波降解法）: we use bioruptor for sonication: 30sec/30sec, the cycles of sonication

dependent on cell type, cell numbers and crosslinking methods. (for example: 293T, 1%formaldehyde, cells did not freeze; 20cyces can obtain acceptable DNA fragment)

7. max speed, 10~15min 4℃ to precipitate cell lysate and aspirate 10ul supernatant for sonication

efficiency test (100-500bp)

8. 95℃,10min to decrosslink protein from DNA fragment , and 1%agarose electrophoretic analysis

(actually, we will get the size from <100bp, or 100-200bp)(Tips: during test, samples should be kept at 4℃ instead of ice bath)

9. If the size of DNA fragment met ChIP grade requirement, then continue to incubation specific antibody

as following; if not, resuspend samples for further sonication

10. Cell lysate should be diluted 10-fold by dilution buffer plus cocktail (150ul cell lysate plus 1350ul dilution

buffer) and left 5ul cell lysate as input.

11. 150ul+1350ul dilution buffer +2.5ug specific antibody (2-5ug), 4℃rotation overnight

Day2: Decrosslinking

Incubating Dynabeads (Dynabeads? Protein G for Immunoprecipitation, 10004D)

12. Add Dynabeads 30ul per sample, 4℃, 3-4hrs Washing beads

13. 1.5ml per sample TSE1 plus 0.5Xcocktail(Roche) ,4℃, rotation 15min~20min 14. 1.5ml per sample TSE2 plus 0.5Xcocktail(Roche), 4℃, rotation 15min~20min

15. 1.0ml TE Buffer wash twice @ R.T, using vacuum to remove supernatant at the first time, and using

ChIP protocol

1ml pipettor to remove TE buffer instead of vacuum because Dynabeads become incompact at the second washing step

Decrosslinking

16. 300ul per sample of TE/SDS buffer (decrosslinking buffer) at 65℃ , 1,400 rpm mixing 1min every 1

hour overnight using Eppendorf Thermomixer. if sample fixed by 1% Glutaraldehyde(irreversible crosslinking), protease K(Ambion, AM2546) should be used to digest first at 50℃ (1,400 rpm mixing 15sec every 30min) in TE/SDS buffer and then 65℃(1,400 rpm mixing 1min every 1 hour) overnight.

Day3: DNA fragment Purification（QIAGEN PCR product purification kit）

17. Quickly short centrifugation and put samples on DynaMag?-2 Magnet(12321D), and transfer

supernatant to a new 2ml tube which contains 1.2ml PB Buffer in advance

18. Mix up and down gently by pipettor, then transfer samples to QIAquick spin column 19. maxi speed, 1 min

20. 750ul PE wash column, maxi speed, 1 min

21. Remove PE, maxi speed 1 min to remove residual ethanol completely 22. 60ul Elution buffer to elute DNA fragment q-PCR test before sequencing

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