The subcellular localization of CmbHLH1 was identified using a transient assay based on onion epidermal cells. The CmbHLH1 open reading frame, lacking its stop codon, was amplified using a Phusion® High-Fidelity PCR kit (New England Biolabs, lpswich, MA, USA) with the primer pair CmbHLH-Dra-F/-Not-R (Table S1 ), and the resulting amplicon was cloned into pMD19-T for sequencing. The same fragment was inserted both into the DraI and NotI cloning sites of the pENTRTM1A dual selection vector (Invitrogen) using T4 DNA ligase (Takara), and into pEarleyGate 10329 (link) using LR ClonaseTM II enzyme mix (Invitrogen). The latter plasmid includes GFP as a marker for transgene expression. The construct was bombarded into the onion cells and GFP expression was monitored by confocal laser microscopy30 .
Pentrtm1a dual selection vector
Manufactured by Thermo Fisher Scientific
Sourced in United States, Japan
The PENTRTM1A dual selection vector is a laboratory tool used for DNA cloning and gene expression. It provides a means for selection and identification of transformed cells. The vector contains genetic elements necessary for replication and antibiotic resistance, enabling researchers to isolate and propagate plasmids containing their genes of interest.
Automatically generated - may contain errors
Lab products found in correlation
T4 dna ligase, by Takara Bio (2 mentions)
Lr clonase 2 enzyme mix, by Thermo Fisher Scientific (2 mentions)
Phusion high fidelity pcr kit, by New England Biolabs (1 mentions)
Gateway technology, by Thermo Fisher Scientific (1 mentions)
Phusion high fidelity dna polymerase, by New England Biolabs (1 mentions)
Gateway system, by Thermo Fisher Scientific (1 mentions)
Pmd19 t, by Thermo Fisher Scientific (1 mentions)
Pmd19 t, by Takara Bio (1 mentions)
3 protocols using pentrtm1a dual selection vector
1
Subcellular Localization of CmbHLH1 in Onion Cells
2
Gateway Cloning for Molecular Studies
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Unless stated otherwise, Phusion High-Fidelity DNA Polymerase (NEB, USA) was used to amplify all DNA sequences using the primer sets listed in S1 Table , and Gateway technology (Invitrogen, USA) was employed to generate plasmids. Coding sequences of HAC1p U, HAC1p S and IRE1p were amplified using yeast cDNA as described above. Coding sequences of bZIP60 U, bZIP60 S, bZIP60ΔN U, bZIP60ΔN S, bZIP60ΔC1, bZIP60ΔC2, IRE1A and IRE1B were amplified using Arabidopsis cDNA (cDNA from DTT-treated seedlings was used for amplification of bZIP60 S and bZIP60ΔN S, whereas cDNA from bzip60-2 seedlings for amplification of bZIP60ΔC2). Coding regions of P1, HC-Pro, P3, 6K1, CI, 6K2, NIaVPg, NIaPro, NIb and CP of TuMV were amplified from the TuMV infectious clone [40 (link)]. With the exception of pENTRTM 1A Dual Selection vector (A10462, Invitrogen) used for IRE1B, all amplified coding sequences were recombined into pDONR221 via the BP reaction (Invitrogen, USA). The entry vector containing P3N-PIPO was described in our previous work [40 (link)]. To highlight the nucleus and to produce donor- and acceptor-only samples (used in FRET assays), constructs bearing 35S::NLS-CFP and 35S::NLS-YFP were created following the BP and LR reactions using the primers listed in S1 Table .
3
Transient Expression of Genes in Onion Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Plasmids for transient expression were generated using the Invitrogen Gateway system. The GUS sequence (without the stop codon) from pBI121, CmNAR2 and CmNRT2 ORFs (lacking their stop codon) were amplified employing primer pairs GUS-BamH I-103/GUS-Xho I-103, NAR2-BamH I-103/NAR2-Not I-103 and NRT2-Sal I-103/NRT2-Not I-103 (Table 1 ), then they were sub-cloned into pMD19-T (TAKARA, Japan) and confirmed by DNA sequencing. The pMD19-T constructs and the pENTRTM1A dual selection vector (Invitrogen) were cut by BamH I/Xho I (GUS), BamH I/Not I (CmNAR2) and Sal I/Not I (CmNRT2), ligated using T4 DNA ligase (TAKARA, Japan), and finally introduced into the pEarleyGate 103 plasmid using an LR Clonase™ II enzyme mix (Invitrogen) to produce pEarleyGate 103-GFP, pEarleyGate 103-CmNAR2/CmNRT2 vector respectively36 (link). The transgenes were transiently expressed in onion epidermal cells using a He-driven PDS-1000 particle accelerator (Bio-Rad). pEarleyGate 103-GFP was used as a control. After bombardment, the onion peels were kept for 24 h in the dark on Murashige and Skoog (1962) medium plates. Confocal laser microscopy was used to monitor the expression of GFP.
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!