Application
ChIP
Sample
Mouse Tissue lysate - nuclear (Brain tissue, whole cortex)
Negative control
During the IP step, I used a general Rabbit IgG as a negative control, which was also used to verify enrichment over input and IgG. I used a gene desert region upstream of Dlx2 as my negative control for my qPCR experiment. Also, a no template control was included to account for technical error (contamination etc).
Specification
Brain tissue, whole cortex
Detection step
Real-time PCR
Type
Cross-linking (X-ChIP)
Duration of cross-linking step: 10 minute(s) and 0 second(s)
Specification of the cross-linking agent: 1% PFA in 1X PBS
Duration of cross-linking step: 10 minute(s) and 0 second(s)
Specification of the cross-linking agent: 1% PFA in 1X PBS
Positive control
I used Grin2b a known direct target of Tbr1 as a positive control for my qPCR experiment to check for enrichment after my IP step.
Other product details
Incubation time
24 hour(s) and 0 minute(s) · Temperature: 4°C · Diluent: please see below
Additional data
Additional Notes
I used Covaris (Duty cycle 5%, Intensity 3, Cycle per burst 20) for 14 cycles. The sonciated chromatin was verified on bioanalyzer and was optimized for a fragment size of 300 bp. following sonication, chromatin was cleared and diluted 1:5 in dilution buffer (dilution buffer consisted of 1.1% Triton X-100, 1.2mM EDTA, 16.7mM Tris.Cl pH=8.0, 167mM NaCl, 0.01% SDS). 200 ul of diluted chromatin was set aside and used as input. I added 8 ug of anti-Tbr1 antibody in 3 mL of the diluted-sonicated chromatin.
Abcam response
Please note, that ChIP is not a guaranateed application. More tests in IP and ChIP are welcomed though.
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.
Dr. Siavash Fazel Darbandi
Verified customer
投稿 Aug 07 2015