EGFP-integrated ROSA26 junction capture PCRs were performed with primers annealing within promoter EF1α (Supplementary Table 1 , Oligo-1) or EGFPSupplementary Table 1 , Oligo-2) and genomic regions past homology sequences (Supplementary Table 1 , Oligos-3, 6). 20 µL reactions contained 0.4 µL Phire II polymerase (Thermo Fisher Scientific), 0.5 µM dNTPS (New England Biolabs), 250 µM primers, 150 ng of template DNA (in vitro) or 250 ng (in vivo). In vitro and in vivo EGFP-integrated junction PCR reactions also contained primers (Supplementary Table , Oligo-4 and -5) to amplify a 616 bp viral sequence to confirm the presence of viral vectors and as a loading control. In vivo, hAAT-integrated ROSA26 junction capture PCR was performed with a primer located within the 3’ end of hAAT (Supplementary Table 1 , Oligo-7) and a primer binding to the 3’ ROSA26 locus, past the right homology sequence (Supplementary Table 1 , Oligo-8). PCR gel images are representative of six independent PCRs and gels.
Phire 2 polymerase
Manufactured by Thermo Fisher Scientific
Phire II polymerase is a high-fidelity DNA polymerase designed for accurate DNA amplification. It offers efficient and reliable performance for a wide range of PCR applications.
Automatically generated - may contain errors
Lab products found in correlation
4 protocols using phire 2 polymerase
1
Targeted EGFP and hAAT Integration PCRs
2
Genotyping Conditional Knockout Mice
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The success of conditional knockout was determined by PCR analysis of DNA extracted from brain tissue where the virus was injected. Genotyping was performed according to the protocol as previously specified (45 (link)). The following primers were used as indicated in Fig. 1B : P1 F: GCC TAG ATT CAC CTG GCT TC; R: GCT CTT AAC CAT TGA GCC ATC T; P1 KO F: CTT GAC CTG TCC CCT TCC CCA TCA AG; R: AGG TTG CAG GGT GGC ATG GCT CTT TTT and Phire II polymerase (Thermo-Scientific #F-170S). PCR was run following the cycling conditions: initial denaturation: 98 °C for 5 min, followed by 98 °C for 5 s, then 10 cycles of 65°C (−0.5°C/cycle) for 5 s, 68°C for 20 s, followed by 30 cycles of 98 °C for 5 s, 60 °C for 5 s, 72 °C for 20 s, followed by a final hold of 72 °C for 1 min. Reactions were separated on 2% agarose gels, yielding the following band sizes: WT, 188 bp; P1, 380 bp; and P1KO, 230 bp.
3
Screening Yeast ade2 Deletion Mutants
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For visual screening of ade2 deletion strains, cloNAT resistant transformants were incubated at 4°C for 7–10 days on YPD agar plates and observed for pink/red colonies. For faster screening, transformants were replica plated onto agar plates with YNB medium (0.17% yeast nitrogen base, 0.5% of (NH4)2SO4, 2% glucose, and 0.079% complete synthetic medium) without adenine, on which C. intermedia ade2 mutants could not grow but turned dark red within 24 h.
For screening of mutants without visible phenotypes, colony PCR was performed. Individual colonies of transformants were lysed in 15 μl deionized water and heated to 98°C for 10 min. PCR analysis was performed using PHIRE II polymerase (Thermo Fisher) with a primer pair designed to anneal upstream and downstream of the homologous flanking regions of the deletion cassette as well as a third control primer that anneal to the target gene. In this way, a successful PCR reaction resulted in either a band of ∼1200 bp identifying true deletion mutants, or a band of ∼1500 bp in false positive transformants where the target gene was still intact.
For screening of mutants without visible phenotypes, colony PCR was performed. Individual colonies of transformants were lysed in 15 μl deionized water and heated to 98°C for 10 min. PCR analysis was performed using PHIRE II polymerase (Thermo Fisher) with a primer pair designed to anneal upstream and downstream of the homologous flanking regions of the deletion cassette as well as a third control primer that anneal to the target gene. In this way, a successful PCR reaction resulted in either a band of ∼1200 bp identifying true deletion mutants, or a band of ∼1500 bp in false positive transformants where the target gene was still intact.
4
Genomic DNA Extraction and PCR Amplification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Ear clips or yolk sacs were lysed either overnight (ear clips) or for 2 hours (yolk sacs) at 55°C in 200 µl of Direct PCR tail lysis buffer (Viagen) supplemented with 200 µg/ml Proteinase K (20 mg/ml stock solution). Proteinase K was inactivated by incubating the samples at 85°C for 15-45 minutes. Samples were cooled to room temperature and spun down (2 minutes at 14,000 rpm), after which 1 µl of the supernatant was used as input for a standard 20 µl PCR reaction with 0.4 µl of Phire II polymerase (ThermoFisher, #F-124S). PCR conditions were as follows: initial denaturation at 98°C for 30 seconds, followed by 30 or 35 cycles of denaturation at 98°C for 5 seconds, annealing at the relevant temperature for 5 seconds, extension at 72°C for 10 seconds, followed by a final extension step of 72°C for 1 minute. Samples were cooled to 16°C and analyzed on a 2% agarose gel in standard TAE buffer. Primer sequences, annealing temperatures, number of cycles and expected band sizes are detailed in Supplementary Table 3.
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!