Resources Search
- Products (57)
- Resources (1836)
Resources
CIP/Lambda Phosphatase Treatment of Cell Lysates:
1) Wash cells 2X with ice-cold PBS.
2) Add cold 1X cell lysis buffer that does not include phosphatase inhibitors [1-2 E7 cells per ml or 0.4ml per 10 cm plate (80-90% confluent)]. Our cell lysis buffer is prepared as follows:
20 mM Tris-HCl (pH 7.5)
150 mM NaCl
1 mM Na2EDTA
1 mM EGTA
1% Triton
1 ug/ml leupeptin
1mM PMSF
3) Add 20ul Quick CIP (NEB #M0525; https://www.neb.com/products/m0525-quick-cip#Product%20Information) per 400ul of cell extract.
4) Incubate with Quick CIP at 37C for 1 hour.
5) Add 20ul lambda protein phosphatase (CST
Resources
When performing CUT&RUN with downstream NG-seq analysis, one can use a normal IgG antibody enriched sample as the negative control. However, we have seen and heard from other scientists that normal IgG antibodies generate very low diversity DNA libraries and can generate what looks to be specific enrichment of genomic regions in the CUT&RUN assay, complicating your NG-seq data analysis. Instead, we recommend generating and using an input chromatin DNA sample as described in the protocol of our CUT&RUN Assay Kit #86652. This input DNA sample generates a more diverse DNA library and works better as a negative control for analysis by NG-seq.