Optogenetic plasmid (pDN34, Addgene plasmid #61343, previously reported in (Niopek et al, 2014 )) containing the LOV2‐Jɑ domain was a gift from Barbara Di Ventura and Roland Eils. Human YAP1‐1δ was PCR amplified from pcDNA3.1 hYAP1‐1δ with primers AH05 and AH06 (listed in Table EV1 ). Phusion High Fidelity DNA polymerase (ThermoScientific) was used for PCR amplification. PCR product and pDN34 were cleaved with KpnI and PmeI FastDigest restriction enzymes (ThermoScientific) for cloning. Optogenetic backbone fused to hYAP1‐1δ is referred to as optoYAP.
Pmei fastdigest restriction enzymes
Manufactured by Thermo Fisher Scientific
PmeI FastDigest restriction enzymes are a type of molecular biology tool used for the cleavage of DNA sequences. They recognize and cleave a specific DNA sequence, enabling precise DNA manipulation and analysis. The core function of these enzymes is to facilitate targeted DNA fragmentation for various research and diagnostic applications.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using pmei fastdigest restriction enzymes
1
Optogenetic Regulation of YAP1-1δ
2
Optogenetic Regulation of YAP1 Localization
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Optogenetic plasmid (pDN34, previously reported in 27 ) containing the LOV2-Jɑ domain was provided by Olivier Destaing. Human YAP1-1d was PCR amplified from pcDNA3.1 hYAP1-1d with primers AH05 and AH06. Phusion High Fidelity DNA polymerase (ThermoScientific) was used for PCR amplification. PCR product and pDN34 were cleaved with KpnI and PmeI FastDigest restriction enzymes (ThermoScientific) for cloning.
Mammalian cell culture and transfection The Nuclear/cytoplasmic ratio was measured using Fiji by drawing a region of interest (ROI) around the cell as well as its nucleus. A macro plugin was used to mask the nucleus from the cytoplasm to calculate the mean intensity in the nucleus and cytoplasm based on the ROI drawn.
Nucleus was demarcated by region without mCherry signal in cells at t = 0 min and region with highest level of mCherry in zebrafish embryos at t = 20 min. Laser power at the focal point was measured using a power meter (Thorlabs).
Mammalian cell culture and transfection The Nuclear/cytoplasmic ratio was measured using Fiji by drawing a region of interest (ROI) around the cell as well as its nucleus. A macro plugin was used to mask the nucleus from the cytoplasm to calculate the mean intensity in the nucleus and cytoplasm based on the ROI drawn.
Nucleus was demarcated by region without mCherry signal in cells at t = 0 min and region with highest level of mCherry in zebrafish embryos at t = 20 min. Laser power at the focal point was measured using a power meter (Thorlabs).
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