To create the MIG (MND Ires GFP)-U2AF1-Flag plasmid, U2AF1-Flag cDNA was amplified from the p3X-Flag-U2AF1 plasmid (obtained from the Kinji Ohno laboratory, Nagoya, Japan) and cloned into the MND-Ires-GFP (MIG) vector.24 (link) The S34F, S34Y, S34F/Q157R, Q157R and Q157P mutations were generated by site-directed mutagenesis (Life Technologies) using the WT MIG-U2AF1-Flag construct as a template. 293T cells were co-transfected with each MIG-U2AF1-Flag expression plasmid described above and either the GH1 or FMR1 minigene reporter constructs described previously.1 (link), 25 (link) GFP+ cells were sorted 48 hours later, followed by RNA extraction and RT-PCR as previously described.1 (link) Amplicons were visualized by polyacrylamide gel electrophoresis and quantified by densitometry (ImageJ). Three independent experiments were performed for each assay and analyzed using the Student’s t-test. Lysates were made from transfected 293T cells and immunoblotting was performed using rabbit polyclonal anti-U2AF1 antibody (Abcam) to confirm over-expression.
Site directed mutagenesis
Manufactured by Thermo Fisher Scientific
Sourced in Japan
Site-directed mutagenesis is a laboratory technique used to introduce specific mutations or changes in the DNA sequence of a gene. It allows for the targeted modification of a DNA molecule to study the impact of specific genetic changes on gene function or to create desired modifications for various applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti u2af1 antibody, by Abcam (2 mentions)
Lysis buffer, by Promega (1 mentions)
Lipofectamine 3000 kit, by Thermo Fisher Scientific (1 mentions)
Pgl3 luciferase reporter vector, by Promega (1 mentions)
Phusion high fidelity dna polymerase, by New England Biolabs (1 mentions)
Glomax 20 20 luminometer, by Promega (1 mentions)
Gateway lr clonase 2, by Thermo Fisher Scientific (1 mentions)
Flag rap1b v12, by Addgene (1 mentions)
Flag rap1b n17, by Addgene (1 mentions)
Clone express 2 one step cloning kit, by Vazyme (1 mentions)
Pcdna3.1 plasmid, by Thermo Fisher Scientific (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
Glomax 96 microplate luminometer, by Promega (1 mentions)
Sap18, by Santa Cruz Biotechnology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
14 protocols using site directed mutagenesis
1
Generating U2AF1 Mutants in MIG Vector
2
Generating U2AF1 Mutants in MIG Vector
3
Luciferase Assay for miR-145 Binding
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The fragments of the 3′-UTR of FTH1P3 were amplified with Phusion® High-Fidelity DNA Polymerase (NEB, Inc.) from human genomic DNA. After gel extraction, the PCR product was cloned into the multiple cloning sites located downstream of the firefly luciferase coding region of the pGL3 Luciferase Reporter Vectors (Promega, USA). The recombinant vector was named pMIR-FTH1P3-wild-type. The mutations in the miR-145 binding sites were introduced using Site-Directed Mutagenesis (Thermo Fisher Scientific, Inc.). The mutation sequence was confirmed by sequencing (Shengong Bio Company). The resulting vector was designated as pMIR-BCYRN-mutant. pMIR-FTH1P3 or pMIR-FTH1P3-mut vector and miR-145 mimic or miR-145 inhibitor were co-transfected into a 24-well plate according to the Lipofectamine 3000 kit (Thermo Fisher Scientific, Inc.). After 36 h, the cells were harvested and lysed (lysis buffer; Promega Corporation). The luciferase reporter gene assay was detected by the GloMax® 20/20 Luminometer (Promega Corporation) according to the instructions.
4
Ras Family Modulation in hESCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To validate the relevance of the Ras family of small GTPases, human RAP2A cDNA was modified to generate a CA (RAP2A-V12) and DN (RAP2A-N17) isoforms using site-directed mutagenesis (Thermo Fischer). The RAP2A isoforms were then inserted into the lentiviral vector pLenti-PGK Neo or Puro DEST by Gateway LR Clonase II (Thermo Fischer). For RAP1B, CA (bovine Flag-RAP1B-V12, Addgene #118323) and DN (bovine Flag-Rap1B-N17, Addgene #118322) were inserted into the same lentiviral vectors as described above for RAP2A. The resulting vectors were used to transduce WT, PB2 KO, and PB2 OE hESCs.
5
Tat101 Flag-tagged Mutagenesis
6
Site-directed Mutagenesis Techniques
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In all cases, mutagenesis was performed by site-directed mutagenesis (Stratagene) or using the Phusion site-directed mutagenesis technique (Thermo).
7
Generation of Influenza Reverse Genetics Plasmids
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The eight gene segments of CK/S1220 and CK/SD098 were inserted into the vRNA-mRNA bidirectional transcription vector pBD with a CloneExpress II one-step cloning kit (product no. C112-02; Vazyme). The protein expression plasmids for PB2, PB1, PA, and NP were generated by inserting the gene segments into the pcDNA3.1(+) plasmid (Invitrogen) with the CloneExpress II one-step cloning kit. Mutations were introduced into the NP gene by site-directed mutagenesis (Invitrogen), according to the manufacturer’s protocol. All of the primer sequences used for the construction of plasmids are shown in Table S2 in the supplemental material. All of the constructs were completely sequenced to ensure the absence of unwanted mutations.
8
Rescuing and Mutating Viral Gene Segments
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Each of the eight SD2/H9N2 viral gene segments was amplified and rescued by using the pBD plasmid as previously described42 (link). Mutations encoding specific amino acid substitutions were introduced using site-directed mutagenesis (Invitrogen, USA). All the constructs were verified by sequencing. The whole sequences of the wild-type SD2/H9N2 virus, HA gene of SD3/H9N2 virus and HA gene of JL3/H9N2 virus were deposited in GenBank under accession numbers KM411625-KM411634.
9
Viral RNP Polymerase Activity Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A dual-luciferase reporter assay system (Promega, Madison, WI, USA) was used to compare the polymerase activities of viral RNP complexes (Xu et al., 2016 (link)). The indicated viruses' PB2, PB1, PA, and NP gene segments were separately cloned into the pCDNA3.1 expression plasmid. Mutations were introduced into the PB1 gene by site-directed mutagenesis (Invitrogen) according to the manufacturer’s protocol. All constructs were sequenced to ensure the absence of unwanted mutations. The primer sequences used for cloning are available upon request. The PB2, PB1, PA, and NP plasmids (125 ng each plasmid) were used to transfect 293T cells with the Luci luciferase reporter plasmid (10 ng) and the renilla internal control plasmid (2.5 ng). Cultures were incubated at 33°C and 37°C. Cell lysates were analyzed 24 hr after transfection to measure firefly and renilla luciferase activities using GloMax 96 microplate luminometer (Promega).
10
Plasmid Construction for Virus Rescue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Construction of plasmids in viral RNA vRNA-mRNA bidirectional expression plasmid pBD for virus rescue was performed as described previously (8 (link)) with the primers shown in Table 1 . The constructs were designated pBD/DK/08-PB2, pBD/DK/08-PB1, pBD/DK/08-PA, pBD/DK/08-HA, pBD/DK/08-NP, pBD/DK/08-NA, pBD/DK/08-M, pBD/DK/08-NS, pBD/CK/09-PB2, pBD/CK/09-PB1, pBD/CK/09-PA, pBD/CK/09-HA, pBD/CK/09-NP, pBD/CK/09-NA, pBD/CK/09-M, and pBD/CK/09-NS, respectively. Mutations were introduced into the PB1 gene by site-directed mutagenesis (Invitrogen) with the primers shown in Table 1 . All of the constructs were completely sequenced to ensure the absence of unwanted mutations.
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!