Round 7 (bead-assisted) and rounds 4, 9, 10, and 11 were amplified in 50 μL PCR reactions containing 0.2 μM template, 0.5 μM primers (forward primer, 5′-CGCATACCAGCTTAGTTCAGAATTCATT-3′; reverse primer, 5′-GCCGAGATTGCACTTACTATCT-3′) and 1× Hot Start Master Mix (10 mM Tris–HCl, 50 mM KCl, 1.5 mM MgCl2, 0.2 mM dNTPs, 5% glycerol, 25 units per mL Hot Start Taq DNA polymerase, pH 8.3) (New England Biolabs). The template was amplified with an initial denaturation at 95 °C for 3 min, 25 cycles of (95 °C for 30 s, 55 °C for 30 s, and 72 °C for 20 s), and a final extension 72 °C for 2 min. The amplified double stranded DNA was purified using a MinElute PCR cleanup column (Qiagen). PCR products were quantified via Nanodrop and normalized to 20 ng μL−1 in nuclease free water. The samples were sent to Genewiz (South Plainfield, NJ) for Amplicon-EZ (150–500 bp) sequencing.
Minelute pcr cleanup column
Manufactured by Qiagen
The MinElute PCR Cleanup column is a laboratory equipment used for the purification of PCR (Polymerase Chain Reaction) products. It is designed to efficiently remove unwanted reagents, primers, and other contaminants from PCR samples, enabling the recovery of purified DNA for further downstream applications.
Automatically generated - may contain errors
7 protocols using minelute pcr cleanup column
1
Amplification and Sequencing of DNA Fragments
2
PCR Amplification and Purification of DNA Library
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The recovered DNA library was amplified in 50 μL PCR reactions containing 0.2 μM template, 0.5 μM primers (forward primer, 5′-/FAM/CGCATACCAGCTTAGTTCAGAATTCATT-3′; reverse primer, 5′-TTTTTTTTTTTTTTTTTTTT/Sp9/GCCGAGATTGCACTTACTATCT-3′) and 1× Hot Start Master Mix (10 mM Tris–HCl, 50 mM KCl, 1.5 mM MgCl2, 0.2 mM dNTPs, 5% Glycerol, 25 units per mL Hot Start Taq DNA polymerase, pH 8.3, New England Biolabs). The template was amplified with an initial denaturation at 95 °C for 3 min, 25 cycles of (95 °C for 30 s, 55 °C for 30 s, and 72 °C for 20 s), and a final extension 72 °C for 2 min. The amplified double stranded DNA was purified using a MinElute PCR cleanup column (Qiagen) and the labeled strand was separated on a denaturing 10% polyacrylamide gel. The desired gel band was excised and >100 picomoles recovered as described previously.
3
EcoRI Digestion of Biosensor Complex
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The biosensor complex was divided into 10 μL portions and 1 μL (5 units) EcoRI (New England Biolabs) was added to each aliquot. The solutions were incubated for 2 h at 37 °C, followed by 20 min at 65 °C to denature EcoRI. The library was recovered using a MinElute PCR cleanup column (Qiagen). Samples were analyzed by denaturing 10% PAGE to monitor digestion of the biosensor. The gels were imaged on a GE Amersham Typhoon RGB scanner using a 488 nm excitation laser and the Cy2 525BP20 emission filter. Digestion efficiency was determined by the percent cleaved (fluorescence band volume for the cleaved product/total lane volume) using ImageJ.
4
Amplification and Purification of Labeled DNA
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The recovered DNA library was amplified in 50 μL PCR reactions containing 0.2 μM template, 0.5 μM primers (forward primer, 5'-/FAM/CGCATACCAGCTTAG-TTCAGAATTCATT-3'; reverse primer, 5'-TTTTTTTTTTTTTTTTTTTT/Sp9/GCCGA-GATTGCACTTACTATCT-3') and 1X Hot Start Master Mix (10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl2, 0.2 mM dNTPs, 5 % Glycerol, 25 units/mL Hot Start Taq DNA polymerase, pH 8.3, New England Biolabs). The template was amplified with an initial denaturation at 95 °C for 3 min, 25 cycles of (95 °C for 30 s, 55 °C for 30 s, and 72 °C for 20 s), and a final extension 72 °C for 2 min. The amplified double stranded DNA was purified using a MinElute PCR cleanup column (Qiagen) and the labeled strand was separated on a denaturing 10 % polyacrylamide gel. The desired gel band was excised and >100 picomoles recovered as described previously.
5
Enzymatic Cleavage Analysis of Biosensor
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The biosensor complex was divided into 10 μL portions and 1 μL (5 units) EcoRI (New England Biolabs) was added to each aliquot. The solutions were incubated for 2 h at 37 °C, followed by 20 min at 65 °C to denature EcoRI. The library was recovered using a MinElute PCR cleanup column (Qiagen). Samples were analyzed by denaturing 10 % PAGE to monitor digestion of the biosensor. The gels were imaged on a GE Amersham Typhoon RGB scanner using a 488 nm excitation laser and the Cy2 525BP20 emission filter. Digestion efficiency was determined by the percent cleaved (fluorescence band volume for the cleaved product/total lane volume) using ImageJ.
6
Enzymatic Cleavage Analysis of Biosensor
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The biosensor complex was divided into 10 μL portions and 1 μL (5 units) EcoRI (New England Biolabs) was added to each aliquot. The solutions were incubated for 2 h at 37 °C, followed by 20 min at 65 °C to denature EcoRI. The library was recovered using a MinElute PCR cleanup column (Qiagen). Samples were analyzed by denaturing 10 % PAGE to monitor digestion of the biosensor. The gels were imaged on a GE Amersham Typhoon RGB scanner using a 488 nm excitation laser and the Cy2 525BP20 emission filter. Digestion efficiency was determined by the percent cleaved (fluorescence band volume for the cleaved product/total lane volume) using ImageJ.
7
Amplicon Sequencing of DNA Fragments
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Round 7 (bead-assisted) and rounds 4, 9, 10, and 11 were amplified in 50 μL PCR reactions containing 0.2 μM template, 0.5 μM primers (forward primer, 5'-CGCATACCAGCTTAG-TTCAGAATTCATT-3'; reverse primer, 5'-GCCGAGATTGCACTTACTATCT-3') and 1X Hot Start Master Mix (10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl2, 0.2 mM dNTPs, 5 % Glycerol, 25 units/mL Hot Start Taq DNA polymerase, pH 8.3). (New England Biolabs). The template was amplified with an initial denaturation at 95 °C for 3 min, 25 cycles of (95 °C for 30 s, 55 °C for 30 s, and 72 °C for 20 s), and a final extension 72 °C for 2 min. The amplified double stranded DNA was purified using a MinElute PCR cleanup column (Qiagen). PCR products were quantified via Nanodrop and normalized to 20 ng/µL in nuclease free water. The samples were sent to Genewiz (South Plainfield, NJ) for Amplicon-EZ (150 -500 bp) sequencing.
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