This experiment introduces the PCR-based DNA library screening method. This experiment is derived from PCR Laboratory Guide (Second Edition) by Seed Kang and Qu Lijia.
Operation method
PCR-based DNA library screening experimental methods
Materials and Instruments
PCR mixtures PCR primer oligonucleotides Hybridized oligonucleotides PCR positive control PCR primary mixtures Distilled water Move I. Materials For more product details, please visit Aladdin Scientific website.
Agarose gel electrophoresis equipment
1. Buffers, solutions and reagents
Distilled water
2. enzymes and enzyme buffers
PCR mixture (can be put together on ice), for lml reaction, including: 200nmol of each dNTP; 1XVent solution or equivalent; 2.5umol of MgCl2; 2nmol of each primer
PCR primary mixture (must be freshly prepared for each reaction): lulVent DNA polymerase or equivalent; 99ul of the above PCR mixture
3. Nucleic acids and oligonucleotides
2 PCR primer oligonucleotides
1 hybridization oligonucleotide
PCR positive control: 10 ng of whole genomic DNA or a starting library component that produces a positive PCR signal (see step 5).
4. culture medium
LB, 1L water: 10 g peptone, 5 g yeast extract, 10 g NaCl, adjust pH to 7.5SM with NaOH, 1L water: 5.8 g NaCl, 2 g MgS04, 50 ml 1mol/L Tris-HCl (pH 7.5), 5 ml 2% gelatin.
LB containing 10 mmol/LMgSO4
5. Special equipment
Agarose gel electrophoresis equipment, including ethidium bromide
6. Other
96-well plate (CorningCostar)
Polyester sealing film (NalgeNuncInternational)
Materials required for phage hybridization
7. Vectors and bacterial strains
DNA libraries constructed with phage vectors
Bacterial strains used for amplification of libraries
II.
Before screening the DNA libraries, several parameters need to be measured to establish effective experimental conditions. These parameters are listed below.
1.PCR conditions. Use primers from the screened library to vary the annealing temperature and the number of cycles to get the maximum amount of specific product. The source of the template can be either the library to be screened ( 106 phage particles) or 1 ng of whole genomic DNA. phage DNA is released to serve as template during the PCR process. Therefore, there is no need to purify the phage DNA prior to the reaction. typical primers that can be used should yield a product of 0.1 to 1.0 kb (16 to 24 nucleotides in length, 50% G+C content). If desired (e.g., when using primers that pair with different exons and span a large intron), the target sequence may be longer than 1 kb, but the amount of product may be reduced.
2. Determine the abundance of genes in the library. Quantify the library by varying the number of phages added, using the PCR conditions established above. The minimum number of phages that can produce a PCR product is the experimentally determined abundance of the gene in the library. For a genomic library with inserts averaging 20 kb, a complexity greater than 105 is required to ensure that the target gene occurs at least once. For a typical library of high complexity, 104 to106 phages are used as templates to show the abundance of genes in the library. The number of phages added that contain one or two copies of the target gene should be used as a library screen.
Once the PCR conditions and abundance of genes have been determined, library screening can be performed as follows.
1. Take 0.5 ml of fresh, overnight cultured bacterial culture in LB, mix it with 0.5 ml of SM, add phage containing the library, and incubate for 20 min at room temperature.
2. Add 20 ml of LB containing 10 mmol/LMgSO4, dispense into 96-well plates according to 8X8 matrix, 100ul per well, seal the plates with polyester sealing film, incubate at 37°C 225r/min for 5-6 h, and amplify the phage. After amplification, the phage concentration should be increased to about 109 phage/ml. about 1/3 of the culture will be equally distributed into the 96-well plate, which should be taken into account when calculating the number of phage added in step 1.
3. Mix the phage in a row or column of 8 wells (25ul per well) with a multiwell pipette (see Variable Patterns in the Discussion section). In this step, be sure to take extra care when removing the sealing film and pipetting to avoid cross-contamination of the samples. If you want to avoid cross-contamination, centrifuge the plate briefly to help remove the liquid from the sealing film. Reclose the plate with polyester sealing film and store at 4°C for up to 1 month.
4. Dilute the phage 1:1 with distilled water and the phage is now ready to be used as a PCR template.
5. Add 0.5ul of template (phage) to 24.5ul of PCR master mix and perform PCR using the PCR conditions established above. 1 negative control (no template) and 1 positive control (i.e., 10ng of total genomic DNA that produces a positive signal or a small portion of the starting library) are required for each experiment.
6. The PCR products are analyzed by agarose gel electrophoresis. The gel is stained with ethidium bromide and photographed (Sambrook and Russell 2001).
7. The gel is dried in vacuo at 70°C, the DNA is denatured, and the oligonucleotide probe (terminally labeled with 32P) is then hybridized directly to the dried gel using standard DNA hybridization conditions, washed, and then radioautographically visualized [technical details of this step can be found in Israel (1993)]. This step is optional when specific PCR products can be easily visualized by ethidium bromide staining, as discussed below. Alternatively, DNA can be transferred to nitrocellulose or nylon membranes using standard techniques and then hybridized.
The data from steps 6 and/or 7 should identify sublibraries containing the target gene (see Discussion section). The first round of screening is now complete, with the positive sublibraries enriched for the gene compared to the starting library, and the subsequent screening cycle is a repetition of steps 1-7, which is carried out after quantification of the amplified sublibraries by phage.
8. The amount of phage in the positive wells was determined by phage spotting (Sambrook and Russell 2001).
9. Start the next round of screening by infiltrating the bacteria with approximately 1/30th of the number of phages from the previous round.
10. Repeat steps 2 to 9.