Ez 10 spin column plasmid dna miniprep kit

Wild-type PTCHD1 cDNA (RefSeq NG_021300) was In-Fusion^TM cloned into a pOPINN vector and transformed into DH5alpha. Positive colonies were selected after O/N culture on Luria Broth (LB) agar plates with ampicillin. The colonies were grown for a further 8 h at 37 °C, 225 rpm in LB with ampicillin before purifying the plasmid using the EZ-10 Spin Column Plasmid DNA Miniprep Kit (Bio Basic, Markham, ON, Canada) according to the manufacturer’s instructions. Successful cloning was confirmed by Sanger sequencing.
A pFastBac^TM vector expression system was then used to produce baculovirus-infected Sf9 cells. PTCHD1-HRV3C-GFP-H6 was PCR-amplified from the pOPINN plasmid, restriction digested and cloned into a pFastBac^TM-CVHG vector. To determine an optimal expression strain, the generated bacmid construct was transformed into two E. coli strains, DH10Bac (Invitrogen, Waltham, MA, USA) and MultiBac^TM EmBacY (Geneva Biotech, Geneva, Switzerland) [45 (link)] and grown O/N at 37 °C, 225 rpm in Luria Broth and subsequently plated on selective LB plates and incubated 24 h at 37 °C. Positive colonies were identified via blue/white screening, expanded, and the bacmid DNA was purified using the EZ-10 Spin Column Plasmid DNA Miniprep Kit (Bio Basic). Successful cloning was confirmed by Sanger sequencing.

Selected oligonucleotides from cycle eight were amplified with the forward and unmodified reverse primer (5′-ATACCAGCTTATTCAATT-3′) and GoTaq® DNA Polymerase (Promega, Madison, USA). The PCR product was cloned into the pGEM®-T easy vector (Promega) and transformed into the E. coli NovaBlue. Fifty colonies were selected, plasmid DNA was purified using EZ-10 spin column plasmid DNA Miniprep Kit (Bio Basic Inc., NY, USA) and the aptamer sequence of each clone determined (Eurofins Genomics, Ebersberg, Germany). DNA sequences were analysed with Clustal Omega⁶⁹ . To keep aptamers short, nine selected aptamer sequences without primer binding regions were synthesised as non-labelled and 5′-biotin-lablled oligonucleotides (Eurofins Genomics).