The experimental material Tricolor tricolor was collected from the wild and established a population in the laboratory.
Experimental procedure
1. Preparation of total DNA of tricolor book lice (1) Place 50 book lice in a 1.5 mL centrifuge tube, use a pestle to quickly grind the test insects in a liquid nitrogen environment, then add 200 μL of DNA extraction lysate.
(2) Add 2.5 μL of proteinase K solution (20 mg / mL) and water bath at 50 ° C for 2 h (or incubate overnight).
(3) Use equal volume of balanced phenol (Tris-HC1 saturated phenol, pH7.8), shake gently (about 20 min), and store at 20 ℃ for 5-10 min.
(4) Centrifuge for 10 min at 4 ° C, and transfer the viscous water phase to a clean centrifuge tube.
(5) Use equal volume of phenol: chloroform (1: 1), phenol: chloroform: isoamyl alcohol (25: 24: 1) and chloroform to extract once, centrifuge (9000 r / min 10 min) to take supernatant .
(6) Add 0.2 times volume of sodium acetate solution (10 mol / L) and twice volume of absolute ethanol, and leave at -20 ℃ for 2h to fully precipitate DNA.
(7) Centrifuge (12000 r / min for 10 min), discard the supernatant, and wash the precipitate twice with 70% ethanol (4 ° C).
(8) After the precipitate is air-dried (to remove ethanol as much as possible), resuspend the DNA in TE buffer (pH 8.0), and gently shake it at 37 ° C to resuspend the DNA and fully dissolve the DNA.
(9) Add Rnase to a final concentration of 1 μg / mL and incubate at 37 ° C for 1 hr.
(10) Press (4) ~ (9) to extract, precipitate, resuspend the DNA, and store in 1μL of double-distilled sterilized water or TE at 4 ℃.
(11) Mix 4μL of DNA with 2μ1 6x loading buffer, apply onto 1.4% agarose gel, and run at 2.5-5 v / cm.
(12) 0.5μg / mL EB staining for 15-30min, observe and take pictures under ultraviolet light.
The concentration of the prepared DNA is detected by the SmartSpecTM3000 nucleic acid concentration analyzer. The cuvette needs to be washed repeatedly with 100 μL / time of sterile water before measuring the absorbance of the water, and then the DNA solution is diluted 50 times for detection. OD = 1.7 is required.
2. Long PCR detection of symbiotic bacteria Wolbachia in Tricolor
The 20 uL reaction system of Long PCR includes:
10 x buffer5 μL
25 mM MgCl2 2 μL
10 mM dNTP 0.7 μL
Pwo enzyme 1 U
Taq DNA5 U
Template 10 ng
10 μM primer 1.6 μL
wsp-F, 5-TGG TCC AAT AAG TGA TGA AAG AAA CTA GCT A
wsp-R, 5-AAAAAT TAAACG CTA CTC CAG CTT CTG CAC
Replenish the reaction system to 20 μL with sterile water
Amplification conditions were: 94 ° C for 2 min, pre-denaturation, 94 ° C for 10 sec, 65 ° C for 30 sec, 68 ° C for 1 min, 10 cycles; 94 ° C for 10 sec, 65 ° C for 30 sec, 68 ° C for 1 min 25 cycles, each cycle was extended at 68 ° C for 20 sec.
After the amplification, 5 μL of the PCR product was detected by 1.5% agarose gel electrophoresis (voltage 5v / cm, electrophoresis buffer was 1 X TAE), and the results were observed under the Bio-Rad gel imaging system.
3. Determination and analysis of sequences
The Wolbachia wsp gene sequence was obtained by direct sequencing of Long PCR product Shanghai Shengong. DNA sequence search and homology comparison use BLAST tool (NCBI website). The maximum likelihood method (ML) in the program is used to reconstruct the phylogenetic tree. The confidence of the bootstrap test of the nodes in the phylogenetic tree is estimated by repeating the calculation 1000 times. Phylogenetic analysis of Wolbachia's wsp gene fragments in the tricolor book lice, and Wolbachia's wsp gene fragments in some other hosts were obtained through GenBank.
HDPE bottles offer stronger tensile strength than LDPE containers, are harder and more opaque, and can withstand somewhat higher temperatures: 120°C (248°F) for short periods, 110°C (230°F) for longer periods. High-density polyethylene cannot withstand autoclaving. HDPE (high density polyethylene) plastic bottles offer a mildly stiff impact resistant bottle, with a great moisture barrier. The HDPE plastic bottles are offered in a great range of sizes, shapes and color.Choose between flip tops, pumps, atomizers or standard lids.
Plastic HDPE Bottle,1 Oz(30ml) Hdpe Plastic Bottle,Hdpe Plastic Bottle,Bottle Opaque Plastic Bottle
NINGBO LONGTERM INTERNATIONAL TRADE CORP.,LIMTED , https://www.nblongterm.com