ITS2‐specific probe was generated by PCR incorporating biotin‐16‐dUTP. ITS2 probe was purified using Zymo research gel purification kit. RNA isolation was performed as described above, and 5‐μg RNA was loaded on a denaturing gel. Gel electrophoresis, transfer, and crosslinking were performed according to NorthernMax manual suggestions. Prehybridization, hybridization with ITS2 probe, washes, blocking, IRDye 800CW streptavidin incubation (1:10,000), and imaging were performed as described and recommended by the Odyssey Infrared Imaging System.
Gel purification kit
Manufactured by Zymo Research
The Gel Purification Kit is a lab equipment product designed to isolate and purify DNA fragments from agarose gels. It provides a simple and efficient method for extracting DNA of interest from electrophoresis gels.
Automatically generated - may contain errors
Lab products found in correlation
T4 dna ligase, by New England Biolabs (2 mentions)
Dna clean concentrator kit, by Zymo Research (2 mentions)
T4 dna ligase buffer 10x, by New England Biolabs (1 mentions)
Qiaprep spin miniprep kit, by Qiagen (1 mentions)
Mix2seq, by Eurofins (1 mentions)
Nebuilder hifi dna assembly mix, by New England Biolabs (1 mentions)
Genejet plasmid miniprep kit, by Thermo Fisher Scientific (1 mentions)
Dna oligo, by Integrated DNA Technologies (1 mentions)
E coli dh5α, by Thermo Fisher Scientific (1 mentions)
Q5 high fidelity dna polymerase, by New England Biolabs (1 mentions)
Miniprep kit, by Qiagen (1 mentions)
Taq dna polymerase, by New England Biolabs (1 mentions)
One shot top10 competent cells, by Thermo Fisher Scientific (1 mentions)
Phusion dna polymerase, by New England Biolabs (1 mentions)
C1000 touch thermal cycler, by Bio-Rad (1 mentions)
Snabi, by New England Biolabs (1 mentions)
Bstbi, by New England Biolabs (1 mentions)
Dh5α competent cells, by Thermo Fisher Scientific (1 mentions)
Sacii, by New England Biolabs (1 mentions)
Dna clean and concentrate kit, by Zymo Research (1 mentions)
5 protocols using gel purification kit
1
ITS2 RNA Northern Blot Analysis
2
Standardized Molecular Biology Protocols
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Unless otherwise specified, all molecular biology protocols were performed using NEB’s High-Fidelity Phusion Master Mix for PCR, Invitrogen’s Gel Purification Kit for gel extraction and Zymo’s DNA Clean & Concentrator kit for DNA purification (5 μg loading capacity). Qiagen’s QIAprep Spin Miniprep Kit was used to isolate plasmid DNA from cell culture. 3’-Azido-3’-Deoxythymidine (Azidothymidine, AZT) was purchased from Sigma-Aldrich. 5’-Triphosphate -3’-Azido-3’-Deoxythymidine (AZT-TP) was obtained through US Biological Life Sciences. All nucleotide oligomers and gBlocks were ordered from Integrated DNA Technologies. Ligations were performed using New England Biolabs’ T4 DNA Ligase Buffer (10x) and T4 DNA Ligase (400,000 units/mL).
3
Cloning and Assembly of Genetic Constructs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PCR was performed using Q5® High-Fidelity DNA Polymerase (M0491, NEB) with DNA oligos from Integrated DNA Technologies (IDT). PCR products were gel purified using Gel Purification Kit (D4002, Zymo Research). To generate entry vectors, DNA fragments were inserted into BsaI-digested GreenGate empty entry vectors [52 (link)] via Gibson assembly (2× NEBuilder Hifi DNA Assembly Mix, NEB) or restriction ligation with T4 DNA ligase (NEB). Base editors, gRNAs, and fluorescent reporter vectors were assembled using Golden Gate cloning (30 cycles (37°C, 5 min; 16°C, 5 min); 50°C for 5 min; 80°C for 5 min) with BsaI or BbsI [52 (link)]. Vectors were transformed by heat-shock transformation into DH5α E.coli or One Shot™ ccdB Survival™ competent cells (Thermo Fisher Scientific). Cells were plated on lysogeny broth medium containing 100 μg mL-1 carbenicillin, 100 μg mL−1 spectinomycin, 25 μg mL−1 kanamycin, or 40 μg mL−1 gentamycin, depending on the selectable marker. Plasmids were isolated (GeneJET Plasmid Miniprep kit, Thermo Fisher Scientific) and confirmed by restriction enzyme digestion and/or Sanger sequencing (Eurofins, Mix2seq). All plasmids are described in Additional file 3 . All primers are listed in Additional file 4 .
4
Construction of Tol2 Transgenic Plasmids
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A HindIII-EGFP-PLCδ-PH-KpnI fragment was PCR amplified and subcloned into pDONR221-MCS-T2A. The resultant pME-EGFP-PLCδ-PH-T2A plasmid was used with p5E-10XUAS (Kwan et al., 2007 (link)), p3E-mRubyCAAX (Kwan et al., 2007 (link)), or p3E-mApple-gg in LR recombination reactions into the destination vector pDestTol2CG. See Supplemental Methods table for primers used.
Competent Escherichia coli was grown in LB broth supplemented with antibiotics under standard conditions. Plasmid DNA was purified using Qiagen Mini Prep kits. For purification of Dsc2l-GFP Bacterial Artificial Chromosome, a NucleoSnap Plasmid Midi kit (Takara Bio) was used. PCRs were carried out using Phusion DNA polymerase or Taq DNA Polymerase and commercial buffers (New England Biolabs). Reactions were run on a BioRad C1000 Touch thermal cycler. PCR products were either cleaned up using DNA Clean & Concentrator kits (Zymo Research) or gel purified using a Gel Purification kit (Zymo Research). Plasmid DNA was transformed into OneShot Top10 competent cells (Invitrogen). Oligonucleotides were synthesized by Integrated DNA Technologies.
Competent Escherichia coli was grown in LB broth supplemented with antibiotics under standard conditions. Plasmid DNA was purified using Qiagen Mini Prep kits. For purification of Dsc2l-GFP Bacterial Artificial Chromosome, a NucleoSnap Plasmid Midi kit (Takara Bio) was used. PCRs were carried out using Phusion DNA polymerase or Taq DNA Polymerase and commercial buffers (New England Biolabs). Reactions were run on a BioRad C1000 Touch thermal cycler. PCR products were either cleaned up using DNA Clean & Concentrator kits (Zymo Research) or gel purified using a Gel Purification kit (Zymo Research). Plasmid DNA was transformed into OneShot Top10 competent cells (Invitrogen). Oligonucleotides were synthesized by Integrated DNA Technologies.
5
Cloning and Expressing GAD65 and H3N2 HA Reactive TCRs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The GAD65 reactive TCR expression construct was described in Ref. [29 (link)]. The sequence for the H3N2 HA reactive TCR HA1.7 was obtained from the NCBI nucleotide database: GenBank accession number X63455.1 (TCRα), and X63456.1 (TCRβ), as deposited by Hewitt et al. [30 (link)]. The variable TCRα and TCRβ chains were ordered as separate gBlocks (IDT), or with both the TCRα and TCRβ sequence in the same gBlock, containing 5′ and 3′ adaptors with respective restriction sites, and sequentially cloned into the TCR-pMSCVII-Ametrine (TCR-pMIA) plasmid [29 (link)]. Briefly, the TCRα gBlock and vector were digested with SnaBI and SacII (both New England Biolabs), and the digested gBlock product was purified with a DNA clean and concentrate kit (Zymo Research), while the digested TCR-pMIA plasmid was run on an agarose gel and the correct band purified with a gel purification kit (Zymo Research). The purified insert and plasmid backbone were ligated, and the product expanded in DH5α competent cells (Invitrogen). The insert was confirmed by sequencing, and subsequently the TCRβ was cloned into the vector by restriction digestion using MfeI and BstbI (both New England Biolabs), using the same setup as for the cloning of the TCRα chain.
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!