核心提示：Day 11. Start 5 ml o/n cultures of strains to be testedDay 21. Inoculate 50 ml of the desired media with a volume of a
Day 11. Start 5 ml o/n cultures of strains to be testedDay 21. Inoculate 50 ml of the desired media with a volume of a saturated o/n culture and grow o/n with shaking at 200 rpm. The volume of cells inoculated should be such that the culture reaches the desired A600 the next morning. Day
- Check A600 until desired cell density is reached 2. Once the desired cell density is reached remove the culture from the shaker3. To the 50 ml culture add 1.39 ml formaldehyde , mix by swirling4. Incubate at room temperature 10-20 min with occasional swirling 5. Add 2.75 ml 2.5 M glycine , mix by swirling6. Incubate for 5 min at room temperature7. Pellet cells in GSA at 5000 rpm for 10 min, decant supernatant8. Wash cells with 20 ml cold TBS+125 mM glycine9. Wash cells with 20 ml cold TBS, decant supernatant10. Resuspend cells in the TBS that remains in the bottle after decanting , and transfer the cells to a Marsh 2.0 ml tube11. Pellet cells in a cold microcentrifuge for 10-30 sec, remove all supernatant12. Freeze cell pellet on dry ice and store at �C70 degrees CDays 3 and 4 are easily combined if the cells are ready early in the day.Day 41. Resuspend cell pellets in 400 ul ChIP lysis buffer+protease inhibitors2. To each sample add an equal volume of glass beads 3. Lock every Marsh tube to ensure the tubes don’t open during lysis4. Lyse cells in the Breeden Lab FastPrep vortex 3 x 40 sec, with 40 sec pauses between runs, at level 4.5 M/s 5. Remove locks and pierce tube bottoms with a hot 20 G 1.5 needle 6. Put pierced tubes into 15 ml Sarstedt tubes and spin in a cold clinical centrifuge at top speed for 1 min 7. Emulsify the insoluble pellets with their supernatants and transfer to 2.0 ml round bottom tubes 8. Sonicate extracts in Breeden Lab water bath sonicator at level 5, 100% duty, 4 x 30 sec with 30 sec pauses on ice between runs 9. Clarify extracts in cold room microcentrifuge for 10 min at top speed10. Transfer supernatants to 1.5 ml tubes and centrifuge again in cold room for 5-10 min at top speed11. Transfer supernatants to 1.5 ml tubes12. Quantitate extracts by Bradford assay: 1 ul of a 1:6 dilution works well in the assay for samples taken from cultures between OD 1 and 213. Freeze extracts on dry ice, and store at �C70 degrees CDay
- Thaw extracts on ice and transfer a volume of extract to a new 1.5 ml tube for IP2. Include necessary controls 3. Bring all 1mg aliquots to 200 ul with ChIP lysis buffer+protease inhibitors, mix well 4. Transfer 2 ul from each 200 ul IP sample to a new 1.5 ml tube with 150 ul ChIP elution buffer and set aside until the IP’s are complete 5. Add Ab to each IP sample except for the no-Ab control, nutate in cold room for 3 hrs6. When there is 30 min left from step 5 prepare rProtein A Sepharose beads &Mac183; Spin down beads and remove supernatant&Mac183; Wash beads twice with 1ml ChIP lysis buffer+protease inhibitors per 250 ul bead volume&Mac183; Resuspend beads after washing in an equal volume of ChIP lysis buffer+protease inhibitors to make 50% slurry 7. Add 60 ul of 50% bead slurry to each IP sample, nutate in cold room 1hr8. Spin beads down in cold room microcentrifuge for 10-15 sec, remove the supernatant, and wash with the following solutions&Mac183; 2x 1ml ChIP lysis buffer+protease inhibitors&Mac183; 2x 1ml ChIP high salt lysis buffer+protease inhibitors&Mac183; 2x 1ml ChIP wash buffer&Mac183; 2x 1ml 1xTE 9. After the last wash draw off as much liquid as possible from the beads using a finely pulled Pasteur pipette10. Resuspend the beads in 85 ul ChIP elution buffer and incubate at 65 degrees C and 950 rpm in a Thermomixer for 10 min11. Spin down beads in microcentrifuge and transfer 75 ul of the supernatant to a new 1.5ml tube 12. Add 75 ul to the beads, repeat steps 10 and 11, and combine the two 75 ul elutions
- For each extract being tested you should now have an IP sample and an IN sample plus any controls14. Incubate all IP and IN samples at 65 degrees C overnight to reverse crosslinksDay 61. Add 750 ul Qiagen buffer PB to each IP and IN sample2. Purify using the Qiagen PCR purification kit, elute DNA in 50 ul buffer EB3. Analyze the samples by quantitative PCR using an appropriate protocol
ChIP Buffers2.5 M glycine 1x TBS + 125 mM glycine 1x TBS ChIP lysis buffer 50 mM HEPES pH 7.5140 mM NaCl1% TritonX-1000.1% Sodium Deoxycholate1mM EDTAChIP lysis buffer 50 mM HEPES pH 7.5500 mM NaCl1% TritonX-1000.1% Sodium Deoxycholate1 mM EDTAChIP wash buffer 10 mM Tris pH 8.0250 mM LiCl0.5% NP-400.5% Sodium Deoxycholate1 mM EDTA1x TE pH 8.0 ChIP elution buffer 50 mM Tris pH 8.01% SDS10 mM EDTAProtease inhibitorsPMSF, Benzamidine, Pepstatin, Leupeptin, and ChymostatinPhosphatase inhibitors Solution A for 10 ml100 mM sodium pyrophosphate 0.45g100 mM sodium orthovanadate 0.18gWater to 10 mlSolution B for 10ml100 mM beta-glycerophosphate 0.29g100 mM EGTA 0.38g1M NaF 0.42gWater to 10 ml
Notes on ChIP method:Day 2Step 1. A dilution of 1:1000 of a saturated culture of SHy278 will grow to approximately OD600 1.0 in 16 hours when grown at 30 degrees C.Day 3Step 4. The duration of formaldehyde treatment is a point of optimization. Shorter treatments will generally yield higher concentrations of soluble protein, but less extensive cross-linking of proteins to DNA. Longer treatments will yield the opposite, and consequently there will be less material in the extracts for doing IP’s. However, this is not the first step that should be optimized. 20 minutes is probably a good starting point for this step unless the effect being observed is expected to occur in a very short time frame. In that case 5-10 minutes formaldehyde treatment may be more appropriate. Optimization of this step involves maximizing the IP/IN ratio for the protein-DNA interaction being assayed, and keeping the duration of formaldehyde treatment short. This can be examined by PCR after IP conditions have been optimized by Western analysis.Day 4Step 1. If assaying for phosphorylated proteins, add phosphatase inhibitors at this and subsequent steps when protease inhibitors are added.Step 4. Pliers are often useful for loosening the knobs on the FasePrep vortex when removing the lysates.Step 8. The water bath sonicator develops a film over the membrane used to sonicate the samples. For best results run the sonicator at level 10 for a few seconds to break the film up before adding samples to the water and sonicating at level 5. It may also be a good idea to add some ice to the water bath to keep the samples as cool as possible.Quality Control: After the extracts are made you can verify efficient shearing of DNA by the following:1. mix 20 ul extract + 1ul 10% SDS + 0.5 ul proteinaseK2. incubate 1hr at 37 degrees C followed by 2 hrs at 65 degrees C3. phenol/chloroform extract the samples4. chloroform extract the samples5. EtOH ppt DNA with 10 ug/ml glycogen 6. resuspend DNA in 20 ul TE7. treat with 40 ug/ml RNAse for 1hr at 37 degrees C8. run half of each sample on a 1.5% agarose gel with EtBr with an appropriate DNA sizing ladder Step 12. ChIP extracts made as described are generally 15-25 mg/mlDay 5Step 5. The amount of Ab used in the assay must be determined empirically. Important considerations include: the fraction of your protein-of-interest the Ab is able to pull down, how much the protein is left after elution, and the ratio of specific PCR signal to noise. Before proceeding to the main experiment it is important to optimize the IP conditions using a combination of Western and PCR analyses to address the aforementioned issues.
ChIP PCR using 32P-dCTP1. Dilute purified DNA samples in watera. Correct dilutions will need to be determined empirically by titration. Reactions should be set up such that the signal for IP and INPUT samples are approximately equal.b. Several dilutions of each sample being assayed should be made, and 10 ul of each dilution used in PCR. Using several dilutions of each sample allows confirmation that the reactions are in the linear range.2. Make a 2x PCR mix for every set of primers being used. For every 20 ul reaction add to the mix the following:a. 2 ul 10x Mg free bufferb. 2 ul 2 mM dNTP mixc. 0.6 ul 50 mM MgCl2d. 1 ul each 20 uM primere. 2.8 ul water f. 0.4 ul Platinum Taqg. 0.2 ul 32P-dCTP3. Add 10 ul PCR mix to each 10 ul DNA sample4. Amplify DNA using the following program:a. 95 degrees 2 minutesb. 95 degrees 30 secondsc. anneal 30 secondsd. 72 degrees 1 minutee. repeat steps b-d 19-25 more times f. 72 degrees 3 minutesg. hold at 4 degrees5. Spin down reactions and add 4 ul 6x loading dye to each6. Run 10 ul of each reaction on 6% PAGEa. use 0.5x TBE as the running bufferb. run the gel at 180 volts for about 75 minutesc. once the bromophenyl blue is 2/3 to 3/4 down the gel stop runningd. some 32P-dCTP will reach the lower buffer, but most will remain in the gel below the BPB if it is not run too longe. cut the gel off just above the BPB and throw the bottom section of gel into the radioactive DSW binf. dry the gel on two pieces of Whatman 3 MM paper