A segment covering bp 123 through 1280 of the mouse Cdkn1b 3’ UTR (NM_009875.4) containing Pumilio-binding elements was subcloned into psiCHECK-2 vector (Promega) by using XhoI and PmeI restriction enzymes (New England Biolabs). Wild-type and mutant PBE sequences were as follows: wt PBE, 5’-TGTATATA-3’; PBE mutant, 5’-acaATATA-3’ (Weidmann and Goldstrohm, 2012 (link)). Mutations were introduced using by ClonExpress MultiS One Step Cloning Kit (Vazyme Biotech). NIH 3T3 cells in 12-well plates were transfected with 100 ng of psi-CHECK-2 construct carrying either wild-type or mutant Cdkn1b 3’ UTR plus 400 ng of CMV-Pum1 or control pCMV using FUGENE HD Transfection Reagent (Promega). After 24 hr, Firefly expression and Renilla lucif-erase expression were measured using the Dual Luciferase Reporter Assay System (Promega) according to the manufacturer’s instructions.
Xhoi and pmei restriction enzymes
Manufactured by New England Biolabs
Sourced in United States
XhoI and PmeI are type II restriction enzymes that recognize and cleave specific DNA sequences. XhoI recognizes and cleaves the palindromic DNA sequence 5'-C^TCGAG-3', while PmeI recognizes and cleaves the palindromic DNA sequence 5'-GTTT^AAAC-3'. These enzymes are commonly used in molecular biology and genetic engineering applications that require the manipulation of DNA molecules.
Automatically generated - may contain errors
Lab products found in correlation
3 protocols using xhoi and pmei restriction enzymes
1
Investigating Cdkn1b 3' UTR Regulation
2
PUM1 regulation of CDKN1B 3' UTR
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CDKN1B 3′ UTR was subcloned into psiCHECK-2 vector (Promega, USA) using XhoI and PmeI restriction enzymes (New England Biolabs, USA). Wild-type PBE sequences 5′-TGTATATA-3′ was mutant to 5′-acaATATA-3′ as previous report[12 (link)]. Cells were co-transfected with pCMV6-hPUM1 and luciferase reporter plasmids, containing fragments or full-length 3′ UTRs. After 48 hours, cells were washed with PBS and lysed in Passive Lysis Buffer (Chroma-Glo Luciferase Assay System, Promega). Then, 20 μL of each lysate was analyzed using the Dual-Luciferase Reporter Assay System (Chroma-Glo Luciferase Assay System, Promega) in a 96 Microplate luminometer (BioTek, USA).
3
Investigating Cdkn1b 3' UTR Regulation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A segment covering bp 123 through 1280 of the mouse Cdkn1b 3’ UTR (NM_009875.4) containing Pumilio-binding elements was subcloned into psiCHECK-2 vector (Promega) by using XhoI and PmeI restriction enzymes (New England Biolabs). Wild-type and mutant PBE sequences were as follows: wt PBE, 5’-TGTATATA-3’; PBE mutant, 5’-acaATATA-3’ (Weidmann and Goldstrohm, 2012 (link)). Mutations were introduced using by ClonExpress MultiS One Step Cloning Kit (Vazyme Biotech). NIH 3T3 cells in 12-well plates were transfected with 100 ng of psi-CHECK-2 construct carrying either wild-type or mutant Cdkn1b 3’ UTR plus 400 ng of CMV-Pum1 or control pCMV using FUGENE HD Transfection Reagent (Promega). After 24 hr, Firefly expression and Renilla lucif-erase expression were measured using the Dual Luciferase Reporter Assay System (Promega) according to the manufacturer’s instructions.
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