PAGE was performed in 1× TBE buffer solution (0.09 M Tris-boric acid and 0.002 M EDTA) with 12% native gels. Oligonucleotides (5 μM) were loaded on the gel, and electrophoresis was run at 64 V for 2 h at room temperature. After electrophoresis, the gel was stained using either 20 μM CyT in Tris-K+ or 1× SYBR Gold, under constant agitation for 15 min, then lightly rinsed with water and visualized using GE Typhoon 9400 Trio. Fluorescence images with excitation wavelength of 532 nm were recorded using the emission filters of 575 nm (for SYBR Gold) and 610 nm (for CyT).
Typhoon 9400 trio
Manufactured by GE Healthcare
The Typhoon 9400 Trio is a high-performance fluorescence and chemiluminescence imaging system designed for life science research laboratories. It is capable of detecting and analyzing a wide range of fluorescent and chemiluminescent samples, including gels, blots, and microplates.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using typhoon 9400 trio
1
Native Gel Electrophoresis of Oligonucleotides
2
RecA-Mediated DNA Strand Exchange Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A three-stranded DNA
exchange reaction was carried out in complete
reaction buffer containing 25 mM Tris-HCl or 25 mM MES, 10 mM magnesium
acetate, 3 mM potassium glutamate, 5% glycerol, 2 mM DTT, 2 mM ATP,
an ATP regeneration system (2 unit/mL pyruvate kinase and 1.5 mM 2-phosphoenolpyruvate),
and 2 mg/mL BSA at a specified pH. For the DNA substrate preference
experiment, 10 nM 500-nt ssDNA or 500-nt Cy3-dsDNA was preincubated
with 1.6 μM RecA (∼0.31 RecA/nt) in complete reaction
buffer for 10 min with or without an additional 5 min of incubation
in the presence of 50 nM E. coli SSB
(Promega, M3011). The reaction was initiated after the addition of
homologous 500-nt Cy3-dsDNA or 500-nt ssDNA and quenched with 40 mM
EDTA, 0.5% SDS and 2 mg/mL proteinase K at a reaction time of 30 min.
The reaction products were checked on 2% agarose gel and monitored
with a Typhoon 9400 & Trio (GE Healthcare). For the pH-dependent
experiment, 30 nM 427-nt ssDNA molecules were preincubated with 4
μM RecA (∼0.31 RecA/nt) in complete reaction buffer.
The experiment was initiated after the addition of homologous 335-nt
dsDNA and quenched with 40 mM EDTA, 0.5% SDS and 2 mg/mL proteinase
K at a reaction time of 30 min. The experiments were performed at
37 °C. The reaction products were checked on 2% agarose gel stained
with GelRed (Biotium) and quantitated with ImageJ.
exchange reaction was carried out in complete
reaction buffer containing 25 mM Tris-HCl or 25 mM MES, 10 mM magnesium
acetate, 3 mM potassium glutamate, 5% glycerol, 2 mM DTT, 2 mM ATP,
an ATP regeneration system (2 unit/mL pyruvate kinase and 1.5 mM 2-phosphoenolpyruvate),
and 2 mg/mL BSA at a specified pH. For the DNA substrate preference
experiment, 10 nM 500-nt ssDNA or 500-nt Cy3-dsDNA was preincubated
with 1.6 μM RecA (∼0.31 RecA/nt) in complete reaction
buffer for 10 min with or without an additional 5 min of incubation
in the presence of 50 nM E. coli SSB
(Promega, M3011). The reaction was initiated after the addition of
homologous 500-nt Cy3-dsDNA or 500-nt ssDNA and quenched with 40 mM
EDTA, 0.5% SDS and 2 mg/mL proteinase K at a reaction time of 30 min.
The reaction products were checked on 2% agarose gel and monitored
with a Typhoon 9400 & Trio (GE Healthcare). For the pH-dependent
experiment, 30 nM 427-nt ssDNA molecules were preincubated with 4
μM RecA (∼0.31 RecA/nt) in complete reaction buffer.
The experiment was initiated after the addition of homologous 335-nt
dsDNA and quenched with 40 mM EDTA, 0.5% SDS and 2 mg/mL proteinase
K at a reaction time of 30 min. The experiments were performed at
37 °C. The reaction products were checked on 2% agarose gel stained
with GelRed (Biotium) and quantitated with ImageJ.
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!