Mutagenesis

Opioid Project Plasmid Information

We purchased plasmids containing protein coding sequences for OPRM1, KCNJ3 and KCNJ9 from purchased from DNASU Plasmid Repository (KCNJ9 ID: HsCD00295074, KCNJ3 ID: HsCD00732503, OPRM1 ID: HsCD00629305). The beginning and end the protein coding sequence of each plasmid was sequenced via Genewiz Sanger sequencing 01-31-19, and the Ape file of each plasmid was updated according to the sequencing results (KCNJ9_pENTR, KCNJ3_pANT7, and OPRM1_pANT7 at the bottom, open with Ape Plasmid Editor). KCNJ3 and OPRM1 did not have stop codons. We performed PCR mutagenesis to add stop codons to KCNJ3 in its original plasmids from ASU. The primers used are shown in magenta in the KCNJ3_pANT7_with_STOP_codon SnapGene file below. Genewiz Sanger Sequencing will be performed to confirm if the KCNJ3 STOP codon was correctly fixed. We will not fix the OPRM1 STOP codon in the pANT7 plasmid from ASU since we will subclone OPRM1 into a pGHE backbone containing a STOP codon. However, we do have primers to fix the OPRM1 STOP codon in pANT7 which are shown in OPRM1_pANT7_with_STOP_codon SnapGene files, but we have not used them.

We are currently working on sticky ended PCR subcloning and HIFI DNA Assembly to transfer the protein coding sequence of KCNJ9, KCNJ3, GRK2, ARRB2, and OPRM1 to the pGHE plasmid. Step by step plans for the proteins not yet completed will be provided on individual pages found under the subcloning link. LH designed HIFI primers (see pGHE+KCNJ9.dna, pGHE+KCNJ3.dna, pGHE+OPRM1.dna, ARRB2+pGHE.dna, and GRK2+pGHE.dna files at the bottom, open with SnapGene) and KCNJ3 and OPRM1 were successfully subcloned in to pGHE (pending sequencing results of end of plasmid). KCNJ9 HIFI subcloning is in progress. Once this process is completed for KCNJ9, it will be began for GRK2 and ARRB2.

Daily updates on what is being done with the Opioid Project should be recorded below on the Google Drive spreadsheet: Opioid Project Mutagenesis Lab Notebook. In addition to this, a link to a table is provided to monitor the progress of the proteins related to this project.

Blunt-end subcloning was attempted but it failed.