Plate-laying and transfer experiments for stickies and plasmid libraries
Plate-laying and transfer experiments for stickies and plasmid libraries
The library should be amplified as soon as possible once it is packaged; it can greatly increase the copy number of the library, but there may be some potential change in the composition of the library during amplification due to the different growth rates of the clones. This change in the library clone composition ratio can be minimized by pre-adsorbing the library clones onto bacteria and using a method of high-density plate laying and short-term incubation.
Operation method
basic program
Materials and Instruments
Plasmid Move 1. On antibiotic-containing plates, determine the titer of the plasmid and mucilage libraries by serial isocratic dilution, calculate the optimal amount of bacterial suspension for spreading the plate and dilute it into 5-10 ml of LB Pepperidge base. For more product details, please visit Aladdin Scientific website.
LB NaOH Chloramphenicol Tris-Cl NaCl
Nitrocellulose Filter Membrane Centrifuge Brinell's Funnel Incubator
2. Prepare a sterile 10 cm or 15 cm layer of Whatman 3 MM filter paper and add 10-20 ml of LB medium to a glass Brinell's funnel or porcelain filter funnel.
3. Place the nitrocellulose filter membrane with marked directions into the LB plate containing antibiotics and move it to the filtering device, then add the bacterial suspension to the filter membrane with a pipette, taking care not to add liquid to the edge of the filter membrane at 4~5 mm.4. Aspirate the liquid slowly, and after the suspension is dried up, remove the filter membrane and transfer it to the plate containing antibiotics, and incubate at 37℃ until colonies of 1~2 mm in diameter grow.
5. Mark and wet another nitrocellulose filter membrane.
6. Place the filter membrane with clones (bacterial side up) on several sheets of 20 cm x 20 cm Whatman 3MM filter paper.
7. Wear gloves and place the wetted nitrocellulose filter membrane over the bacterial membrane in the marked position, preferably staggered by 2-3 mm between the membranes to facilitate separation between the membranes.
8. The clones were transferred by covering the nitrocellulose membrane with three 20 cm x 20 cm sheets of 3 MM filter paper and pressing a glass plate of the same size onto it.9. Remove the glass plate and filter paper pressed against the filter membrane and use a 20-G needle to puncture a small hole in the overlapping original and transfer membranes to mark the orientation.
10. They are then separated and placed in agar plates with the colonized side facing up for overnight growth, with the transfer membranes incubated at 37°C and the original filter membranes incubated at 25°C.
11. Store the master agar plate at 4 °C or continue with the transfer experiment.
12. Transfer the filter membrane to LB plates containing 50 μg/ml chloramphenicol and incubate at 37s°C for 4~10 h to amplify mucoids and plasmids.13. Next, the filter membrane was placed, transfer side up, on three 46 cm x 75 cm 3MM Whatman filter papers saturated with each of the following three liquids for 5 min.
(1) 0.5 mol/l NaOH
(2) 1 mol/l Tris-Cl, pH 7.5
(3) 1 mol/l Tris-Cl, pH 7.5 / 1.25 mol/l NaCl
14. The filter membrane was blotted dry with 3 MM filter paper and then dry baked in a vacuum oven at 80°C for 90 min.
15. Hybridization experiments were carried out using incision shift labeled probes with the filter membrane.