Plate laying and transfer experiments of phage libraries
Plate laying and transfer experiments of phage 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
Phage Move 1. Titration of phage libraries by serial isocratic dilution.2. The recombinant phage was mixed with the plate-laying host bacteria in culture tubes (Table I) and incubated at 37 ℃ for 20 min. For more product details, please visit Aladdin Scientific website.
Agarose LB NaOH SSC Tris-Cl
Nitrocellulose membrane Incubator
Table I. Optimal preparation method of phage library plate-laying components3. Determine the number of plates that will be used for library screening by the number of phages per plate.
4. The number depends on the number of recombinant phages in the library and the probability of occurrence of the target clone.
5. Add 0.7% top agarose to the test tube and quickly pour it onto the LB top plate held at 37 ℃ and incubate it at 37 ℃ for about 6~12 h until the phage spots are all over the plate but not connected to each other with appropriate size.
6. The plate was placed at 4 ℃ for more than 1 h before it was used for membrane photocopying experiments.7. Mark the nitrocellulose filter membrane with the original ballpoint pen, marking side up.
8. Cover the filter membrane with a pre-cooled LB plate with phage spots for 1~10 min after marking the direction of localization by puncturing with a 20-G needle.
9. Remove the membrane with flat-tip tweezers and gently place it face up on a paper towel or filter paper, and allow it to dry for more than 10 min at room temperature.10. Next, the membrane was placed, transfer side up, on three Whatman 3 MM filter papers saturated with each of the following three liquids for 1 to 2 minutes each.
(1) 0.2 mol/l NaOH / 1.5 mol/l NaCl
(2) 0.4 mol/l Tris-Cl pH 7.6 / 2×SSC
(3) 2×SSC10. Place the membrane in a vacuum oven at 80℃ for 90~120 min, or in a constant temperature box at 42℃ overnight, if the hybridization experiment is not done for the time being, the membrane can be clamped with dry filter paper and stored at room temperature.