DNA was extracted from 100 mg of brain tissue by using a DNeasy Blood and Tissue Kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions. The PRNP coding sequence was amplified in a 50 µL final volume by using 5 µL of extracted DNA, eluate 1X AmpliTaq Gold 360 PCR Buffer (Life Technologies, Carlsbad, CA, USA ), 2.5 mmol/L MgCl2, 1X 360 GC Enhancer (Life Technologies), 200 µmol/L dNTPs, 0.25 µmol/L of forward (5′-GCTGACACCCTCTTTATTTTGCAG-3′) and reverse (5′-GATTAAGAAGATAATGAAAACAGGAAG −3′) primers (11 (link)), and 0.5 µL AmpliTaq Gold 360 (Life Technologies), according to the following amplification protocol: 5 min at 96°C; 30 s at 96°C; 15 s at 57°C; 90 s at 72°C for 40 cycles; and 4 min at 72°C. Amplicons were purified with the Illustra ExoProStar 1-Step clean-up kit (GE Healthcare Life Sciences, Little Chalfont, UK), sequenced using the Big Dye Terminator Cycle Sequencing Kit v1.1 (Life Technologies), and purified with the Big Dye XTerminator Purification Kit (Life Technologies), and detected by using an ABI PRISM 3130 apparatus (Life Technologies).
Illustra exoprostar 1 step clean up kit
Manufactured by GE Healthcare
Sourced in United Kingdom
The Illustra ExoProStar 1-Step clean-up kit is a laboratory equipment product designed to purify nucleic acid samples. It is a simple and efficient method for removing contaminants and inhibitors from DNA or RNA samples prior to downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Bigdye xterminator purification kit, by Thermo Fisher Scientific (2 mentions)
Amplitaq gold 360, by Thermo Fisher Scientific (2 mentions)
Abi prism 3130apparatus, by Thermo Fisher Scientific (2 mentions)
Dneasy blood and tissue kit, by Qiagen (2 mentions)
Bigdye terminator cycle sequencing kit v1, by Thermo Fisher Scientific (1 mentions)
Bigdye terminator v1.1 cycle sequencing kit, by Thermo Fisher Scientific (1 mentions)
Seqscape version 2, by Thermo Fisher Scientific (1 mentions)
2 protocols using illustra exoprostar 1 step clean up kit
1
Brain PRNP Gene Sequencing Protocol
2
Prion Protein Gene Amplification and Sequencing
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We extracted DNA from 100 mg of frozen brain tissue with DNeasy Blood and Tissue
Kit (QIAGEN, Hilden, Germany) following the manufacturer’s instructions.
We amplified the PrP gene (PRNP) coding sequence in a
50-μL final volume using 5 μL of extracted DNA, 1× AmpliTaq
Gold 360 PCR Buffer (Applied Biosystems, Foster City, CA, USA), 2.5 mmol/L
MgCl2, 1× 360 GC Enhancer, 200 μmol/L dNTPs, 0.25 μmol/L of
forward (5′-GCTGACACCCTCTTTATTTTGCAG-3′) and reverse
(5′-GATTAAGAAGATAATGAAAACAGGAAG-3′) primers (19 (link)), and 0.5 μL of AmpliTaq Gold
360 (Applied Biosystems), according to the following amplification protocol: 5
min at 96°C; 30 s at 96°C, 15 s at 57°C, 90 s at
72°C for 40 cycles, and 4 min at 72°C.
We purified amplicons by using an Illustra ExoProStar 1-Step clean-up kit (GE
Healthcare Life Sciences, Little Chalfont, UK). We conducted sequencing
reactions by using the BigDye Terminator v1.1 Cycle Sequencing Kit, purified
using BigDye XTerminator Purification Kit, and detected with the ABI PRISM 3130
apparatus (all Applied Biosystems). We analyzed sequences by using Seq Scape
version 2.5 (Applied Biosystems).
Kit (QIAGEN, Hilden, Germany) following the manufacturer’s instructions.
We amplified the PrP gene (PRNP) coding sequence in a
50-μL final volume using 5 μL of extracted DNA, 1× AmpliTaq
Gold 360 PCR Buffer (Applied Biosystems, Foster City, CA, USA), 2.5 mmol/L
MgCl2, 1× 360 GC Enhancer, 200 μmol/L dNTPs, 0.25 μmol/L of
forward (5′-GCTGACACCCTCTTTATTTTGCAG-3′) and reverse
(5′-GATTAAGAAGATAATGAAAACAGGAAG-3′) primers (19 (link)), and 0.5 μL of AmpliTaq Gold
360 (Applied Biosystems), according to the following amplification protocol: 5
min at 96°C; 30 s at 96°C, 15 s at 57°C, 90 s at
72°C for 40 cycles, and 4 min at 72°C.
We purified amplicons by using an Illustra ExoProStar 1-Step clean-up kit (GE
Healthcare Life Sciences, Little Chalfont, UK). We conducted sequencing
reactions by using the BigDye Terminator v1.1 Cycle Sequencing Kit, purified
using BigDye XTerminator Purification Kit, and detected with the ABI PRISM 3130
apparatus (all Applied Biosystems). We analyzed sequences by using Seq Scape
version 2.5 (Applied Biosystems).
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