PCR ribotyping was performed using capillary gel electrophoresis as previously described.7 (link) The 16S rRNA gene primers were labeled with carboxyfluorescein. PCR products were analyzed in the ABI 3100 genetic analyzer (Applied Biosystems, Foster City, CA, USA) with 36-cm capillary loaded with a POP4 gel (Applied Biosystems). A Size Standard-1200 bp TAMRA ladder (Chimerx, Milwaukee, WI, USA) was used as internal size marker for each sample. The peak Scanner software 1.0 (Applied Biosystems) was used to determine the size of each peak. The data obtained were submitted to the WEBRIBO database (https://webribo.ages.at/
Peak scanner software 1
Manufactured by Thermo Fisher Scientific
Sourced in United States
Peak Scanner Software 1.0 is a data analysis software tool designed for researchers working with chromatographic data. The software provides a platform to visualize, analyze, and process peak information from a variety of chromatographic techniques.
Automatically generated - may contain errors
Lab products found in correlation
4 protocols using peak scanner software 1
1
PCR Ribotyping using Capillary Electrophoresis
2
Molecular Fingerprinting of Candida glabrata
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Six short tandem repeat markers described for C. glabrata (ERG3, MTI, RPM2, GLM4, GLM5, and GLM6) (Foulet et al., 2005 (link); Abbes et al., 2012 (link)) were amplified by PCR for all isolates using forward labeled primers as previously described (Foulet et al., 2005 (link); Abbes et al., 2012 (link); Duran-Valle et al., 2012 (link)) with the following modifications: GLM5 was labeled with HEX and GLM6 with NED fluorochromes. PCR reaction mixtures contained 20 ng of DNA, 0.2 μM of deoxynucleoside triphosphate, 2 μL of PCR 10× buffer, 2.5 mM of MgCl2, 5 U of Taq DNA, 0.25 μM of RPM2 and ERG3 primers and 1 μM of MTI, GLM4, GLM5, and GLM6 primers in a final volume of 20 μL. PCR program used from amplifying all markers consisted on an initial step of 5 min at 95°C, followed by 40 cycles of 95°C for 30 s, 57°C for 1 min and 72°C for 1 min, and an additional step of 7 min at 72°C. Amplicons were sized by capillary electrophoresis using Hi-Di formamide (Applied Biosystems, Foster City, CA, United States) and ROX 500 (Applied Biosystems, Foster City, CA, United States) as internal size standard, as described (Duran-Valle et al., 2012 (link)). Reactions were analyzed in duplicate, and fragment sizes were calculated using Peak Scanner software 1.0 (Applied Biosystems, Foster City, CA, United States).
3
Optimizing Blocking Primer Effectiveness
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We applied fragment analysis to test and compare the effectiveness of our blocking primers35 (link). Fragment analysis of fluorescently labeled PCR products on capillary electrophoresis can separate fragments in different sizes and can be used as a semi-quantitative method. When amplified with the universal LCO1490 and HCO2198 primers, the expected lengths of PCR products were different for amphipod hosts and Rickettsia COI, because Rickettsial COI is 6 bp longer. The FAM dye was attached to the 5′ end of the LCO1490 primer. This fluorescently labeled forward primer was added to the PCR mixture instead of the normal (unlabeled) LCO1490 primer. Various factors can affect PCR success with blocking primers: Tm of primers, the concentration of primers (relative ratio between blocking primer and regular primer), the amount of the DNA templates in a PCR mixture (concentration of DNA), and the number of PCR cycles35 (link). To optimize PCR conditions, PCR reactions were conducted under several different PCR conditions (Table 1 ). Fragment analyses were carried out with a 1,200 LIZ size marker on an ABI 3730xl System (Applied Biosystems) at Macrogen (Korea). Results were analyzed with Peak Scanner Software 1.0 (Applied Biosystems; https://www.thermofisher.com/ ).
4
Optimizing Blocking Primer Performance
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We applied fragment analysis to test and compare the effectiveness of our blocking primers (Vestheim and Jarman, 2008) (link). Fragment analysis of fluorescently labeled PCR products on capillary electrophoresis can separate fragments in different sizes and can be used as a semiquantitative method. When amplified with the universal LCO1490 and HCO2198 primers, the expected lengths of PCR products were different for amphipod hosts and Rickettsia COI, because Rickettsial COI is 6 bp longer. The FAM dye was attached to the 5' end of the LCO1490 primer. This fluorescently labeled forward primer was added to the PCR mixture instead of the normal (unlabeled) LCO1490 primer. Various factors can affect PCR success with blocking primers: Tm of primers, the concentration of primers (relative ratio between blocking primer and regular primer), the amount of the DNA templates in a PCR mixture (concentration of DNA), and the number of PCR cycles (Vestheim and Jarman, 2008) (link). To optimize PCR conditions, PCR reactions were conducted under several different PCR conditions (Table 2). Fragment analyses were carried out with a 1,200 LIZ size marker on an ABI 3730xl System (Applied Biosystems) at Macrogen (Korea). Results were analyzed with Peak Scanner Software 1.0 (Applied Biosystems).
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!