The vector for CHC2-YFP was constructed as follows: Arabidopsis genomic DNA was used as a template with primers CHC2-Pentr-F and CHC2-Pentr-R to amplify the whole sequence of CHC2. The final PCR product was cloned into pENTR/DTOPO (Life Technologies), and then into the destination vector pEarleyGate 101 by LR reactions (Life Technologies). For the YFP-FLOT1 construct, the full-length CDS of FLOT1 was cloned into the pENTR vector (Life Technologies), then into the destination vector pEARLYGATE 104 for YFP tagging (Life Technologies). For the TET8-CFP construct, the full-length CDS of TET8 was cloned into the pENTR vector (Life Technologies), then into the destination vector pEARLYGATE 102 for CFP tagging (Life Technologies). For the construct to express YFP, the YFP sequence was amplified from pEARLYGATE 101 and then cloned into pENTR vector and finally into pEARLYGATE 100 (Life Technologies). Primer sequences are listed in Supplementary Data 1 .
Pearleygate101
Manufactured by Thermo Fisher Scientific
The PEarleyGate101 is a lab equipment product manufactured by Thermo Fisher Scientific. It is designed to perform a core function, but a detailed description while maintaining an unbiased and factual approach is not available.
Automatically generated - may contain errors
Lab products found in correlation
Pearlygate 104, by Thermo Fisher Scientific (2 mentions)
Pearlygate 102, by Thermo Fisher Scientific (2 mentions)
Pearlygate 101, by Thermo Fisher Scientific (2 mentions)
Pearlygate 100, by Thermo Fisher Scientific (2 mentions)
Pearleygate104, by Thermo Fisher Scientific (2 mentions)
Pentr d topo, by Thermo Fisher Scientific (1 mentions)
Pentr vector, by Thermo Fisher Scientific (1 mentions)
Pentr sd d topo, by Thermo Fisher Scientific (1 mentions)
Pentr sd d topo vector, by Thermo Fisher Scientific (1 mentions)
Lr recombination reaction, by Thermo Fisher Scientific (1 mentions)
Fluoview fv1000 confocal laser scanning microscope, by Olympus (1 mentions)
In fusion system, by Takara Bio (1 mentions)
Gateway system, by Thermo Fisher Scientific (1 mentions)
Pds 100 he particle delivery system, by Bio-Rad (1 mentions)
Gateway technology, by Thermo Fisher Scientific (1 mentions)
Tcs sp2, by Leica camera (1 mentions)
6 protocols using pearleygate101
1
Construction of Fluorescently-Tagged Protein Vectors
2
Cloning Fluorescently Tagged Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The vector for CHC2-YFP was constructed as follows: Arabidopsis genomic DNA was used as a template with primers CHC2-Pentr-F and CHC2-Pentr-R to amplify the whole sequence of CHC2. The final PCR product was cloned into pENTR/SD/D-TOPO (Life Technologies), and then into the destination vector pEarleyGate 101 by LR reactions (Life Technologies). For the YFP-FLOT1 construct, the full-length CDS of FLOT1 was cloned into the pENTR/SD/D-TOPO vector (Life Technologies), then into the destination vector pEARLYGATE 104 for YFP tagging (Life Technologies). For the TET8-CFP construct, the full-length CDS of TET8 was cloned into the pENTR/SD/D-TOPO vector (Life Technologies), then into the destination vector pEARLYGATE 102 for CFP tagging (Life Technologies). For the construct to express YFP, the YFP sequence was amplified from pEARLYGATE 101 and then cloned into pENTR/SD/D-TOPO vector and finally into pEARLYGATE 100 (Life Technologies). Primer sequences are listed in Supplementary Data 1 .
3
Localization of MlGT43A-G Proteins in Tobacco Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The co-localization of fluorescent protein-tagged MlGT43A-G with the Golgi marker was examined using the tobacco leaf transient expression system [58 (link)]. The full-length MlGT43 genes without a terminator codon were amplified and fused with yellow fluorescent protein (YFP) in pEarleyGate101 vector [59 (link)] via LR recombination reactions (Invitrogen). The proteins generated thus encode fusion proteins of MlGT43s with YFP tagged at the C terminus. After 3 days post co-infiltration of YFP fusion proteins and the Golgi marker into tobacco leaves, leaf epidermal cells were examined for yellow fluorescence signal using a FluoView FV1000 Laser Scanning confocal microscope (OLYMPUS) equipped with 488 nm argon laser.
4
Constructing Overexpression, CRISPR/Cas9, and Localization Vectors
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For the construction of overexpression vectors, the CDS sequences amplified from Ws82 cDNA were first cloned into the pDONR-Zeo vector and then cloned into the pEarleyGate101 or pEarleyGate104 vector using the Gateway recombinant system by BP reaction and LR reaction, respectively (Invitrogen) (Earley et al., 2006 (link)). For the construction of CRISPR/Cas9 vectors, at least three gRNA target sites were selected for each gene using the website tool CRISPRdirect2 (Naito et al., 2015 (link)). The soybean hair root system was used to test the efficiency of each gRNA, and then the effective gRNA was selected to construct the single or double knockout vectors (Li et al., 2020 (link); Lyu et al., 2021 (link)). For the construction of subcellular localization vectors, the CDS of the indicted gene was cloned into the pA7-YFP or pA7-RFP vector using the In-fusion system (Clontech). The CDS of GmMYB29 was cloned into the pA7-RFP vector as a nuclear marker (Chu et al., 2017 (link)). The pA7-YFP or pA7-RFP empty vector was used as a control. To construct vectors for yeast two-hybrid experiment, the CDS of the indicated gene was cloned into the pGADT7 or pBridge vector using the In-fusion system, respectively.
5
GmPRR3b Transformation and Mutant Generation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To construct the H6-YFP and YFP-H6 plant transformation plasmids, the coding DNA sequence (CDS) of GmPRR3b H6 (1878 bp) was amplified from the cDNA of young leaves of Williams 82 using primers Attb-H6-F and Attb-H6-R and inserted into the overexpression vector pEarleyGate 101 or pEarleyGate 104 using the Gateway system following the manufacturer's instructions (Invitrogen). To generate the CRISPR/Cas9engineered Gmprr3b mutant, gRNAs were designed using the CRISPR-P website (http://crispr.hzau.edu.cn). The 19-bp DNA fragment coding for the gRNA was inserted into the pCas9-AtU6-sgRNA plasmid at the XbalI site. A CRISPR/Cas9 expression vector with the Cas9 CDS driven by Arabidopsis RPS5A promoter and the customized gRNA transcribed by the Arabidopsis U6 promoter (Tsutsui and Higashiyama, 2017) was constructed. The above expression plasmids were individually introduced into Agrobacterium tumefaciens strain EHA105 via electroporation and then transformed into WT soybean (Tianlong 1, E1e2E3E4 and GmPRR3b H6 background) using the cotyledon-node method (Paz et al., 2006) .
6
Subcellular Localization of BcSnRK2 Genes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To investigate the subcellular localizations of BcSnRK2 genes, two expression vectors were constructed using a transient expression system in onion epidermal cells. The full-length coding sequences of BcSnRK2.6a were amplified using Gateway-specific primers cloned into an entry vector, and then subcloned into pEarleyGate101 using Gateway technology (Invitrogen, Carlsbad, CA). The yellow fluorescent marker protein (YFP) was fused to BcSnRK2.6a. Gold particles with a diameter of 1 μm coated with 35S BcSnRK2.6a-YFP was introduced into onion epidermal cells using particle bombardment (PDS-100/He particle delivery system; Bio-Rad, Hercules, CA). After incubation at 22°C for at least 12 h under darkness, fluorescence and bright-light images were observed using laser scanning confocal microscopy (Leica, TCS SP2, Wetzlar, Germany).
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!