42. To each tailed sample, 90 µl of poly(A) PCR buffer was added to a final concentration of 1x ThermoPol buffer (New England Biolabs), 2.5 mM MgSO4, 1 mM dNTPs (Roche), 100 µg/ml BSA (Roche), 3.75 U Taq polymerase (NEB) and 1.5 U Phusion (NEB) and 2.5 ...
Constructs. RBM10-EGFP was generated by amplifying RBM10 from pFRT-TO-RBM1026, followed by dou- ble digestion of the PCR Product with NheI and EcoRI (NEB) and ligation into NheI/EcoRI linearized pEGFP-N3 vector (Clontech) using T4 DNA ligase (NEB). The tet-on lentiviral plasmids ...
DNA was released from chromatin by adding 5 μL of cleanup buffer (900mM NaCl, 300 mM EDTA, 1.1% SDS, 4.4 mg/mL Proteinase K (NEB)) followed by an incubation for 30 min at 40°C. Tagmentated DNA was isolated using 2 × volumes of SPRI beads and eluted in 21 μl. For library...
All PCR reactions were performed with the Phusion polymerase (NEB, Frankfurt, Germany) according to the manufacturer’s instructions. The vectors pECYFP and pEGFP were made compatible to the Gateway system (Invitrogen) by insertion of the Gateway cassette reading frame B into the Sma I site of ...
BLOCK 1 ;CARD ADDRESS WITHIN INPUT BUFFER .RDNBR:! BLOCK 1 ;NUMBER OF CARDS IN THE BUFFER. .RDSTR:! BLOCK 1 ;READER STREAM NUMBER .RDBFR:! BLOCK 1 ;READER BUFFER ADDRESS. .RDSTA:! BLOCK 1 ;DEVICE STATUS WORD .RDTIM:! BLOCK 1 ;JOB START TIME .RDINI:! BLOCK 1 ;END RDR ...
Standard cloning methods are used in the construction ofPseudomonas fluorescens(Pf) expression plasmids engineered to produce full-length DIG-10 proteins encoded by plant-optimized coding regions. Restriction endonucleases are obtained from New England BioLabs (NEB; Ipswich, Mass.) and T4 DNA Ligase (...
Phage libraries (Ph.D.-12 or Ph.D.-C7C) were diluted to 4 × 1010 pfu/ml from the original library (New England Biolabs, NEB), added to the plate, followed by washing with TBS-T and elution of VirB10-binding phage using 100 μl of elution buffer (0.2 M glycine–HCl, pH 2.1;...
Baselines are adjusted to zero for all curves and double-referenced by subtracting a sensorgram of buffer injected over the coated surface from the experimental sensograms to give curves representing specific binding Curves are modeled assuming a simple 1:1 interaction to generate the equilibrium and...
Cell pellets were harvested by by resuspension in a pre-chilled buffer (5 mL/mg), and cells ruptured using a high pressure homogenizer at single pass of 15000 psi. Protease inhibitor cocktail tablet (1/10 mL) was added, then centrifuged at a maximum speed (16000 × g) at...
RBM10-EGFP was generated by amplifyingRBM10from pFRT-TO-RBM1026, followed by double digestion of the PCR Product withNheI andEcoRI (NEB) and ligation intoNheI/EcoRI linearized pEGFP-N3 vector (Clontech) using T4 DNA ligase (NEB). The tet-on lentiviral plasmids carryingRBM10-EGFP were con...