dsDNA and NCPs containing a single 8-oxodGuo/C base pair (referred to as dsDNA-8-oxodGuo and NCP-8-oxodGuo, respectively) at three different positions (73, 89 and 137) were prepared according to the procedure we reported previously (23 (link)). dsDNA and NCPs containing a single AP at position 137 (referred to as dsDNA-AP137 and NCP-AP137, respectively) were obtained by in situ photolysis of dsDNA and NCPs containing a photoprotected AP137 (21 (link),24 (link)). Unless otherwise specified, dsDNA and NCPs were labelled with fluorescein amidite (FAM) at the 5′ end of the 8-oxodGuo-modified strand. dsDNA and NCPs with a FAM label only on the 3′ end are referred to as dsDNA-8-oxodGuo-3′-FAM and NCP-8-oxodGuo-3′-FAM, respectively. The four histone mutants (H4-del 1−20, H3-del 1−37, H2A-del 1−15, H2B-del 1−31) that we used to prepare tailless NCPs were obtained from Professor Marc Greenberg (Johns Hopkins University). The plasmid used for hOGG1 expression was provided by Professor Bjørn Dalhus (Oslo University Hospital), and hOGG1 was expressed and purified as previously reported (25 (link)). APE1 and proteinase K were purchased from NEB (catalogue nos. M0282S and P8107S, respectively). All reactions were carried out in siliconized tubes. Gels were visualized with an Amersham Typhoon Gel and Blot Imaging System at excitation and emission wavelengths of 488 and 526 nm, respectively.
Typhoon gel
Manufactured by Cytiva
The Typhoon Gel is a high-performance fluorescence and phosphor imaging system designed for gel analysis. It offers high-resolution imaging capabilities for a variety of gel-based applications, including DNA, RNA, and protein gels.
Automatically generated - may contain errors
3 protocols using typhoon gel
1
Preparation and Characterization of Oxidative DNA Lesion-Containing Nucleosomes
2
Preparation and Characterization of DNA-Histone Complexes
3
Cardiac Splicing Profile Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was isolated from frozen ventricular tissue according to the manufacturer’s instructions (TriZol; Molecular Research Center). Complementary DNA was synthesized using SuperScript II RT (Invitrogen) with oligo(dt) primers. Splicing of Sorbs1, Tnnt2, Scn5a, and Cacna1s were analyzed by RT-PCR using the following primers flanking the alternatively spliced exon of interest: Sorbs1: (forward [fwd]) 5′-FAM-ACCTTCCAGCTCAGCTTCCAC-3′ and (reverse [rev]) 5′-ACTTCTCATCCACCTGTCGGAG-3′;Tnnt2: (fwd) 5′-AGCCGAGAGCATGTCTGACG-3′ and (rev) 5′-FAM-TCTGTCTCAGCCTCACCCTCAG-3′;Scn5a: (fwd) 5′-FAM-CTTCTGCCTGCATGCGTTCAC-3′ and (rev) 5′-GGCCTCCACGGAACCATT-3′;Cacna1s: (fwd) 5′-GCTATTTTGGAGATCCTTGGAA-3′ and (rev) 5′-FAM-AGCAGGGTGCGCACACCCTC-3′.
RT-PCR products were resolved on agarose gels and analyzed using an Amersham Typhoon Gel and Bolt Imaging system. Relative band intensities were measured by densitometry analysis using ImageQuant TL. To measure the relative level of exon 6a inclusion in Scn5a, amplified PCR products were visualized on a 2% agarose TBE gel alongside an aliquot of PCR product digested to completion with restriction enzyme Apa1 (16 h at 37°C) in order to cleave any product containing exon 6B.
RT-PCR products were resolved on agarose gels and analyzed using an Amersham Typhoon Gel and Bolt Imaging system. Relative band intensities were measured by densitometry analysis using ImageQuant TL. To measure the relative level of exon 6a inclusion in Scn5a, amplified PCR products were visualized on a 2% agarose TBE gel alongside an aliquot of PCR product digested to completion with restriction enzyme Apa1 (16 h at 37°C) in order to cleave any product containing exon 6B.
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!