A kind of chromatin fragmentation method and its application
A fragmentation method and chromatin technology, applied in the chromatin fragmentation method and its application field, can solve the problems of affecting the specific binding of antibodies and proteins, mixing fragments of different sizes, and narrow size range, so as to avoid the problem of specificity of enzyme cleavage sequences. , Broad application prospects and market value, the effect of reducing the probability of protein denaturation
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Embodiment 1
[0068] 1. Inoculate cells on a 15cm cell plate. When the cell density grows to 60%-80%, suck up the medium to the remaining 15mL, add 1mL of 16% formaldehyde to make the final concentration of formaldehyde 1%, fix at room temperature for 10 minutes, then add 950μL The concentration is 2.5M glycine to terminate the action of formaldehyde, room temperature for 10 minutes;
[0069] 2. After discarding the medium, wash twice with cold 10mL PBS, scrape off the cells and put them into a 1.5mL EP tube, centrifuge at 250g at 4°C for 2min to remove the supernatant, record the cell volume, and add 5 times the cell volume of buffer Resuspend A, centrifuge at 250g for 2 minutes at 4°C to remove the supernatant, add 2 times the cell volume of buffer CF1 to resuspend, centrifuge at 500g for 2 minutes to remove the supernatant, add 1 times the cell volume of buffer CF2 to resuspend, and static on ice Place for 10 minutes, centrifuge at 500g for 2 minutes to remove the supernatant, then add 1...
Embodiment 2
[0078]1. Inoculate cells on a 15cm cell plate. When the cell density grows to 60%-80%, suck up the medium to the remaining 15mL, add 1mL of 16% formaldehyde to make the final concentration of formaldehyde 1%, fix at room temperature for 10 minutes, then add 950μL The concentration is 2.5M glycine to terminate the action of formaldehyde, room temperature for 10 minutes;
[0079] 2. After discarding the medium, wash twice with cold 10mL PBS, scrape off the cells and put them into a 1.5mLEP tube, centrifuge at 250g at 4°C for 2min to remove the supernatant, record the cell volume, and add 5 times the volume of the cell in buffer A Resuspend, centrifuge at 250g for 2min at 4°C to remove the supernatant, add 2 times the cell volume of buffer CF1 to resuspend, centrifuge at 500g for 2min to remove the supernatant, add 1 times the cell volume of buffer CF2 to resuspend, and place on ice 10min, centrifuge at 500g for 2min to remove the supernatant, then add 1mL of buffer N1 to resuspe...
Embodiment 3
[0087] 1. Inoculate cells on a 15cm cell plate. When the cell density grows to 60%-80%, suck up the medium to the remaining 15mL, add 1mL of 16% formaldehyde to make the final concentration of formaldehyde 1%, fix at room temperature for 10 minutes, then add 950μL The concentration of glycine is 2.5M to terminate the action of formaldehyde, room temperature for 10 minutes.
[0088] 2. After discarding the medium, wash twice with cold 10mL PBS, scrape off the cells and put them into a 1.5mLEP tube, centrifuge at 250g at 4°C for 2min to remove the supernatant, record the cell volume, and add 5 times the volume of the cell in buffer A Resuspend, centrifuge at 250g for 2min at 4°C to remove the supernatant, add 2 times the cell volume of buffer CF1 to resuspend, centrifuge at 500g for 2min to remove the supernatant, add 1 times the cell volume of buffer CF2 to resuspend, and place on ice 10min, centrifuge at 500g for 2min to remove the supernatant, then add 1mL of buffer N1 to res...
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