Embedded Files
A plasmid containing a b2AR coding sequence was purchased from DNASU Plasmid Repository (ID HsCD00305465), and the coding sequence was transferred to pGHE (b2AR_pGHE.ape at the bottom). This transfer was achieved using HiFi DNA Assembly with 20 bp overlap. See final plasmid pGHE b2AR plasmid.
G:\Physiology Lab\Norimatsu Lab\Lab Projects\TAAR1\B2AR\DNA\Plasmids\pGHE
G:\Physiology Lab\Norimatsu Lab\Lab Projects\TAAR1\B2AR\DNA\Plasmids\pGHE
b2AR HiFi Primers (Forward and Reverse)
Name (F/R) Oligo (Uppercase = target-specific primer) Len % GC Tm Ta *
Insert_F attccccgggATGGGGCAACCCGGGAAC 24 75 71°C 67°C
Name (F/R) Oligo (Uppercase = target-specific primer) Len % GC Tm Ta *
Insert_R gacccttctaCAGCAGTGAGTCATTTGTAC 26 42 61°C 61°C
pGHE HiFi Primers (Forward and Reverse)
Name (F/R) Oligo (Uppercase = target-specific primer) Len % GC Tm Ta *
Vector_F ctcactgctgTAGAAGGGTCAAGACAATTC 26 46 59°C 59°C
Name (F/R) Oligo (Uppercase = target-specific primer) Len % GC Tm Ta *
Vector_R gttgccccatCCCGGGGAATTGATCTGCC 25 64 68°C 69°C
Left: The 20 bp overlap (highlighted in blue and written in purple) between the b2AR HiFi Insert Reverse Primer and the pGHE (TAAR1) HiFi Forward primer. This is at the 3' end of the b2AR coding sequence.
Left: The 20 bp overlap (highlighted in blue and written in purple) between the b2AR HiFi Insert Forward Primer and the pGHE (TAAR1) HiFi Reverse Primer. This is at the 5' end of the b2AR coding sequence.
Insert primers were run on b2AR in pANT7 (http://dnasu.org/DNASU/GetCloneDetail.do?cloneid=305465)
Vector primers were run on pGHE
Primer design is the most important step of this process. The NEBuilding assembly tool was used to accomplish this. Before primers were created snap gene was used to create the desired final plasmid, b2AR, pGHE. This new plasmid was used to create primers for the pGHE plasmid as well as the b2AR coding sequence.
pGHE primers were then used to amplify the pGHE region of the TAAR1 pGHE plasmid. b2AR primers were used to amplify the b2AR protein coding sequence from the b2AR pANT7 plamid. A gel was ran to verify PCR reaction. DnpI digest was performed on the resulting PCR products, afterwards the two PCR were combined together into a single reaction with the HIFI master mix. A gel was ran to confirm product.
NEBuilder HiFi DNA Assembly Master Mix (PDF)
Site-directed mutagenesis using NEBuilder HiFi DNA Assembly Master Mix (PDF)
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