To confirm that PCR products were of the cyp51A promoter region of A. fumigatus with or without tandem repeats, amplicons from the three A. fumigatus control isolates (1205/wt, 1467/TR34, and 1470/TR46) produced by the TR34-PCR and TR46-PCR assays were sequenced. Additionally, amplicons from environmental samples of 8 TR-positive and 4 non-TR A. fumigatus from the TR34-PCR assay and 4 wt positive from the TR46-PCR assay were sequenced. Amplicons were cut from agarose gels with a sterile scalpel and purified using the Qiagen II gel extraction kit (QIAGEN). Bidirectional Sanger sequencing of the amplification products using the internal primers of each PCR through the chain termination method was performed by GENEWIZ. The sequences were aligned and edited manually using Geneious Prime 2019.2.3 (San Diego, CA, USA). A multiple alignment was performed to observe the presence or absence of the tandem repeats. The Basic Local Alignment Search Tool (http://blast.ncbi.nlm.nih.gov/Blast.cgi ) was used to verify that the sequenced PCR products matched A. fumigatus.
Ii gel extraction kit
Manufactured by Qiagen
The QIAGEN II Gel Extraction Kit is a laboratory tool designed to extract and purify DNA fragments from agarose gels. It facilitates the recovery of DNA fragments after electrophoresis for subsequent applications.
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Lab products found in correlation
3 protocols using ii gel extraction kit
1
Sequencing of cyp51A Promoter Regions
2
PCR Amplification and DNA Extraction
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PCR assays were carried out in the reaction mixture (10 μL) containing 1.5 units rTaq DNA polymerase, 10 pmol each of forward and reverse primer pair, 2.5 nmol each dNTP mixture in PCR buffer (10 nmol Tris-HCl; pH 8.3, 50 nmol KCl, and 1.5 nmol MgCl2) (Takara Bio Inc.). The amplifications were performed with 25 cycles of denaturation at 96°C for 10 s, annealing at 58°C for 20 s, and extension at 72°C for 75 s. The PCR products were analyzed by electrophoresis in 1% agarose gels containing ethidium bromide with 100-bp ladder marker (Takara Bio Inc.). The DNA fragments were excised from the gel and eluted into 10 mM Tris-HCl buffer (pH8.0) containing 1 mM EDTA by using QIAGEN II Gel Extraction Kit (QIAGEN).
3
Optimized PCR Amplification and Purification
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PCR assays were carried out in the reaction mixture (10 µL) containing 1.5 units rTaq DNA polymerase, 10 pmol each of forward and reverse primer pair, 2.5 nmol each dNTP mixture in PCR buffer (10 nmol Tris-HCl; pH 8.3, 50 nmol KCl, and 1.5 nmol MgCl2) (Takara Bio Inc.). The amplifications were performed with 25 cycles of denaturation at 96°C for 10 s, annealing at 58°C for 20 s, and extension at 72°C for 75 s.
The PCR products were analyzed by electrophoresis in 1% agarose gels containing ethidium bromide with 100-bp ladder marker (Takara Bio Inc.). The DNA fragments were excised from the gel and eluted into 10 mM Tris-HCl buffer (pH8.0) containing 1 mM EDTA by using QIAGEN II Gel Extraction Kit (QIAGEN).
The PCR products were analyzed by electrophoresis in 1% agarose gels containing ethidium bromide with 100-bp ladder marker (Takara Bio Inc.). The DNA fragments were excised from the gel and eluted into 10 mM Tris-HCl buffer (pH8.0) containing 1 mM EDTA by using QIAGEN II Gel Extraction Kit (QIAGEN).
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