The experiment was performed as described (27 ) with the following modifications. Nuclei were treated with 1000U EcoRI (NEB, #R3101S) at 37°C overnight. 100U T4 enzyme (NEB, #M0202S) was added to digested nuclei and incubated at 16°C for 4 hours. Another 100U T4 enzyme was added and nuclei were incubated with rotation at 4°C overnight. 150ng of ligated DNA was quantified in triplicate by TaqMan real-time PCR using the PrimeTime Gene Expression Supermix (IDT, #1055772). Control 3C template was generated by using two bacterial artificial chromosomes (BACs) encompassing the entire EGFR gene, RP11-159M24 and RP11-148P17, were purchased from the Children’s Hospital Oakland Research Institute (CHORI). Equimolar of the two BACs were digested with EcoRI and ligated. The ligation product from BAC control was used for normalization. The relative interaction frequency was calculated as: 2Ct (BAC)-Ct (3C).
T4 enzyme
Manufactured by New England Biolabs
The T4 enzyme is a DNA ligase derived from the T4 bacteriophage. It catalyzes the formation of phosphodiester bonds between adjacent 3'-hydroxyl and 5'-phosphate termini in double-stranded DNA, RNA, or mixed DNA-RNA molecules.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using t4 enzyme
1
3C Assay Protocol for EGFR Gene
2
Chromatin Interaction Mapping via 3C
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The experiment was performed as described [52 (link)]. Briefly, cells were crosslinked with 1% formaldehyde at room temperature for 10 min, quenched with 0.125 M glycine, lysed, and treated with 600 U HindIII (NEB, #R3104) at 37 °C overnight followed by a 4 h ligation with T4 enzyme (NEB, M0202L) at 16 °C. Ligated products were quantified in triplicate by TaqMan real-time PCR. Probes and primers (listed in Supplementary Table 2 ) were designed by using primer blast provided by NCBI. Control 3C template was generated by using two bacterial artificial chromosomes (BACs), 656G14 and 1125P18, which together encompass putative K-M enhancer and MGMT promoter regions. Equimolar of the two BACs were digested with HindIII and ligated. The ligation product from BAC control was used for normalization. The relative interaction frequency was calculated as:
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!