3/14/2023 0 Comments Dark noise quality control test![]() For each condition 2500–7000 cells were analyzed. For each cell the mean fluorescence intensity was determined from the segmented area and the background fluorescence subtracted. Segmentation performance was manually inspected. Brightfield images were segmented using a customized program QFTrack written in MatlabR2011b. Images were binned 2 6 2 using SoftWoRx 5.5 software. Samples were imaged with an UPlanSApo 100 6 /1.40na oil objective (Olympus, Tokyo, Japan) with the following exposure settings and filter sets (excitation, emission): GFP: 0.2 s with 100% excitation (475 nm/ 28, 525 nm/50), YFP: 1 s exposure with 100% excitation (513 nm/17, 559 nm/38) and CFP: 2 s exposure with 100% excitation (438 nm/24, 470 nm/24). Brightfield and fluorescence images were taken on a DeltaVi- sion Elite Imaging System (Applied Precision, Issaquah, WA, USA) equipped with Olympus IX71 microscope, a solid state illumination unit and a CoolSNAP HQ2 camera. Samples were sealed with Baysilone high viscose silicone paste (Bayer, Leverkusen, Germany) and covered by a coverslip. Samples were resuspended to a final OD 600nm of 2 and 2 m l were spread on a gel pad made of the same medium solidified with 1.5% (w/v) Ultra Pure Agarose (Invitrogen). Cells were harvested and washed once in M9 medium supplemented with 10 m g/mL erythromycin for B. Strains were cultivated to an OD 600nm of 0.3, 3 and 5 ( B. Strains were inoculated from an LB overnight culture into a 20 mL LB culture without antibiotics with a starting optical density at 600 nm (OD 600nm ) of 0.02. For bglS locus integration cells were transformed with BamHI linearized DNA and checked by colony PCR with primers ST16 and LA38. For Bacillus strains containing amyE integrated reporters two independent clones were stored and used in this study. Finally, the absence of single cross-over events was checked by PCR with primers ST129 and ST130 on genomic DNA. ![]() Clones were verified by sequencing PCR products obtained from genomic DNA with primers ST39 and ST40. Transformants were screened for construct integration into the amyE locus by lack of amylase activity on LB plates containing 1% (w/v) starch followed by treatment with Lugol’s solution. subtilis was transformed using a standard method. ![]() Transformants were checked by colony PCR and sequenced using primers ST16 and ST17. After T4 DNA polymerase treatment, 5 fmol of the vector were annealed with the insert in a molar ratio 1:5 for 10 min at room temperature and transformed into E.coli. Samples were incubated for 20 min at 22 u C and inactivated for 30 min at 75 u C. For the LIC procedure, 40 fmol of the vectors were treated with 0.6 U of T4 DNA polymerase in presence of 2.5 mM dATP and 200 fmol of the promoter construct (708 bp) were treated with 0.6 U of T4 DNA polymerase in presence of 2.5 mM dTTP. The plasmids pGFPamy and pGFP_Star were cleaved with SmaI prior to T4 DNA polymerase treatment. The upstream and downstream parts were fused by overlap extension PCR.
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