Team:CAU China/Protocol



Metarhizium acridum experiment



Medium preparation

10g/L glucose, 2.5g/L Tryptone, 5.0g/L Yeast Extract

pH=6.0

To make a solid 1/4SDAY medium, add agar 20g/L

5g/L NaCl, 5g/L Yeast Extract, 10g/L Tryptone

5g/L NaCl, 5g/L Yeast Extract, 10g/L Tryptone

To make a solid LB medium, add agar 15g/L

Add Salt Czapek Dox Agar medium 109g/L

Metarhizium acridum experiment

Incubated M.acridum in 28℃. To get the spore, incubated for 7~14 days and scrape the spore on the medium. Suspended spore in the 0.05% Tween80 solution.
We use Fungi Genomic DNA Extraction Kit from Solarbio company

1.Take 50~100mg hyphae add 200μL solution A, 20μL RNaseA and 100mg glass beads. Placing on high speed-oscillator for 30min.
2.Adding 20μL protease K（10mg/L）mixing and digesting in 55℃ water pot for 30min. 12000rpm centrifuge for 2min. Taking supernatant to a new EP tube.
3.Adding solution B in supernatant. Placing in 55℃ water pot for 5min if precipitation.
4.Adding 200μL waterless ethanol. Then adding the solution and precipitation into absorption column. Waiting for 2min.
5.12000rpm centrifuge for 1min and remove solution.
6.Adding 600μL PW 12000rpm centrifuge for 1min. Then remove the solution.
7. Adding 600μL PW 12000rpm centrifuge for 1min. Then remove the solution.
8.12000rpm centrifuge for 2min. Placing column in RT or 50℃ oven for a while.
9.Puting column on a new EP tube and add 50~200μL EB，placing for 5min and 12000rpm centrifuge for 1min.
10. Adding the EB solution of the former step in the column again. Placing for 2min and 12000rpm centrifuge for 1min.
1.Competent cell producing
Suspended spore in DEPC water, and then adjust it into spore with 109/mL taking 50μL.
2.Electrotransformation
Taking 5μL linear plasmid and incubated in the ice. Mixing with 50μL suspended spore solution and then add the mix into electrotransformation cup. With 1.2kV, 2.5μF hit, add 1mL YED medium and put it into 1.5mL EP tube, incubated for 15min in the ice. Then incubated in 28℃, 150rpm oscillation cultivate for 90min. Then put the transformation solution on the screening solid medium.
Collect M. acridum spore on the 1/4SDAY and suspended into suspension spore solution with cottonseed oil. Adjust spore with the final concentration of 5X108/mL. Adding 3μL suspension spore solution on locust back plate. The locust added with cotton seed will be the control group. All the locust will be divided into two groups and each group will have three parallel samples with 8 locusts in each sample.
Incubate in 28℃ and observe each sample and calculate the LT50 every 12h. Analyzing the survival result of each group.

Yeast Protocol



Medium Preparation

Yeast extract 10 g/L,
tryptone 20 g/L
glucose 20 g/L,
AGAR 20 g/L,
distilled water.
Stored at 4 ℃
Yeast extract 5g/L
Tryptone 10g/L
NaCl 10g/L
AGAR 20g/L
Stored at 4 ℃
carbon source is replaced by galactose 20g/L. Other components are the same as YPD medium. Stored at 4 ℃
The amino acids in bacterial culture medium are removed, add the yeast carbon source 6.7g/L, glucose 20g/L, complete nutritional mixture 2g/L, agar 20g/L, distilled water.
Stored at 4℃
Add Nutrient deficiency mixture(lack in one or several nutrition) instead of complete nutritional mixture. Other components are the same as SC medium.
Stored at 4℃
The complete nutritional mixture:
Adenine 0.5g
Alanine 2.0g
Arginine 2.0g
Asparaginate 2.0g
Aspartic acid 2.0g
Cysteine 2.0g
Glutamine 2.0g
Leucine 10.0g
Methionine 2.0g
Para aminobenzoic acid 0.2g
Phenylalanine 2.0g
Proline 2.0g
Serine 2.0g
Glutamic acid 2.0g
Glycine 2.0g
Histiding 2.0g
Inosito 2.0g
Isoleucine 2.0g
Threonie 2.0g
Typtophan 2.0g
Tyrpsine 2.0g
Uracil 2.0g
Valine 2.0g

Synthesis of dsRNA in vitro

A T7 RNA polymerase promoter can be added to any DNA sequence using PCR
by including the T7 promoter sequence at the 5′-end of either of the amplification primers.
The minimal T7 RNA polymerase promoter sequence requirement is: 5′-TAATACGACTCACTATAGGN(17–22)-3′.
The +1 base in bold (G) is the first base incorporated into the RNA strand, and the second G should then be followed by 17–22 gene-specific nucleotides. The addition of extra bases upstream of the minimal T7 RNA polymerase promoter (boldface) sequence may increase yield by allowing more efficient polymerase binding and initiation (i.e., 5′ GGATCC-TAATACGACTCACTATAGGN(17-22) 3′). Thus use of the extended T7 promoter sequence is recommended. Generating the necessary two DNA templates requires four PCR primers and two PCR amplifications (see Figure 1). Amplification strategies using primers containing T7 promoter sequences may include an initial 5–10 cycles at an annealing temperature approximately 5°C above the melting temperature of the gene-specific sequences, followed by 20–35 cycles of annealing approximately 5°C above the melting temperature of the entire primer, including the T7 promoter.

PCR products should be examined by agarose gel electrophoresis before transcription to verify that a single PCR product of the expected size is generated. We recommend purifying the PCR product using the Wizard® SV Gel and PCR Clean-Up System (Cat.# A9281), followed by quantitation by absorbance at 260nm. This system allows for both purification and concentration, and if necessary, gel purification of PCR products. Small volumes of unpurified PCR product may be used as template DNA (1–2μl per 20μl reaction), but in general, higher yields are obtained with purified products.
The linear control DNA supplied with the system produces single-stranded transcripts that are 1.1kb and 2.3kb in length. The transcripts produced from the pGEM? Express Positive Control Template are not complementary and thus will not form double-stranded RNA. They are to be used as a positive control for transcriptional activity of the system and RNA integrity following transcription.
1. Set up the appropriate reaction size at room temperature. The 20μl reaction may be scaled as necessary (up to 500μl total volume; use multiple tubes for reaction volumes >500μl). Add the components in the order shown.
T7 Reaction Components sample reaction control reaction
RiboMAXTM Express T7 2X Buffer* 10.0μl 10.0μl
liner Template DNA(`1μg total) 1`8μl 10.0μl
pGEM® Express Positive Control Template 1.0μl
Nuclease-Free Water 0`7μl 7.0μl
Enzyme Mix,T7 Express 2.0μl 2.0μl
Final Volume 20.0μl 20.0μl

*Frozen RiboMAX™ Express T7 2X Buffer will contain a precipitate that can be dissolved by warming the buffer at 37°C and mixing well. Note that the buffer contains spermidine, which can precipitate DNA at temperatures colder than room temperature.
2. Mix gently and incubate at 37°C for 30 minutes (see Notes 1–3).
Notes:
1) For plasmid templates, up to 3μg of purified template may be included per 20μl reaction.
2) To maximize yield, incubation at 37°C may proceed for up to 2–6 hours. In general, however, no dramatic increase in yield is observed beyond the 30-minute incubation period, except for smaller transcripts (~200 bases). A time-course experiment may be performed to determine the optimal incubation time for maximal RNA synthesis.
3) Incubation at 42°C may improve the yield of dsRNA for transcripts that contain secondary structure. If sufficient yield is not obtained at 37°C and the template is GC-rich, incubate the reaction at 42°C instead.
The DNA template can be removed by digestion with DNase following the transcription reaction. RQ1 RNase-Free DNase (Cat.# M6101) has been tested for its ability to degrade DNA while maintaining the integrity of RNA. If accurate RNA concentration determination is desired, the RNA should be DNase-treated and purified to remove potentially inhibitory or interfering components.
1. To anneal the RNA strands, mix equal volumes of complementary RNA reactions together and incubate at 70°C for 10 minutes, then slowly cool to room temperature (~20 minutes). This allows annealing of the doublestranded RNA.
2. Dilute the supplied RNase Solution 1:200 by adding 1μl RNase Solution to 199μl Nuclease-Free Water. Add 1μl freshly diluted RNase Solution and 1μl RQ1 RNase-Free DNase per 20μl reaction volume, and incubate for 30 minutes at 37°C. This will remove any remaining single-stranded RNA and the template DNA, leaving double-stranded RNA. Do not save or reuse the diluted RNase Solution.
1. Add 0.1 volume of 3M Sodium Acetate (pH 5.2) and 1 volume of isopropanol or 2.5 volumes of 95% ethanol. Mix and place on ice for 5 minutes. The reaction will appear cloudy at this stage. Spin at top speed in a microcentrifuge for 10 minutes.
2. A white pellet should be visible at the bottom of the microcentrifuge tube. Carefully pour off or aspirate the supernatant, and wash the pellet with 0.5ml of cold 70% ethanol, removing all ethanol following the wash. Air-dry the pellet for 15 minutes at room temperature, and resuspend the RNA sample in Nuclease-Free Water in a volume 2–5 times the original reaction volume (at least 2 volumes are required for adequate resuspension). Store at –20°C or –70°C. Do not over-dry the RNA pellet, as it may become difficult to resuspend completely.
3. The dsRNA may be further purified following precipitation using a G25 micro spin column following the manufacturer's instructions (Amersham Biosciences, Cat.# 27-5325-01). This will remove any remaining rNTPs and allow accurate quantitation by absorbance at 260nm. We do not recommend processing more than an initial 40μl reaction volume per spin column. A loss of yield can be expected following G25 purification (approximately 66% recovery).

1) Design primers and PCR amplified the target fragment while adding the homologous arm sequence at both ends
2) The vector is linearizeby restriction enzyme digestion. The target fragment and the linearized vector are purified by RealTimes ® agarose gel recovery kit
3) NovoRec recombinase reaction
Reaction system 10μL:
0.5 μL NovoRec recombinase
I μL,10X reaction buffer
Target fragment and linearized carrier
Fill with ddH2O to 10 μL
Reaction at room temperature for 30min.
4) product is transformed into the target competent recipient E.coli cells.
Add 3 μL reaction product in 100 μL competent recipient E.coli cells.
Ice bath, 20 min, 42 ℃ heat shock, 90 s,
Ice bath , 2 min,
add 1 mL fresh LB medium
37 ℃ incubating for 30 min,
Coat on the solid LB medium with corresponding antibiotic, 37 ℃ cultivate for 12 to 20 h.
5) Single colony are selected and cultivated on a new solid medium
6) Colony PCR is used to identify whether the fragment is inserted into the vector.
7) The right colonies are enlarge cultured, and the target plasmid is purified and then sequenced.
1) The yeasts are cultured at 30℃ to OD600=1.0.
2) 1-2 mL culture solution are collected into a 1.5ml centrifuge tube
3000 g, 30 s centrifuge, discard the supernatant.
Repeat once or twice
3) The cells are suspended in 1 mL sterile ultra-pure water, and then centrifuge at 3000 g, 30 s to remove the supernatant.
4) The cells are suspended in 1 mL, 0.1 M LiAc solution.
Centrifuge at 3000 g , 30 s and remove the supernatant.
5) 1 mL single-stranded DNA sample is boiled for 5 min and cooled in ice water rapidly.
6) The invert solution is added to the cells after centrifugation.
The invert solution is composed as follows and added in the following order:
PEG (50% W/V) 240 mu L
1 M LiAc 36 I,
Single stranded DNA (2 mg/mL) 50 L
DNA x L to be transformed
Sterile ultrapure water 34-x L
Total volume 360 L
7) Shake violently until the whole system is completely mixed and place on the ice for 3 min.
8) Heat shock at 42℃ for 40 min.
9) Cells are collected by centrifugation at 2500 g for 30 s, and the supernatant is discarded.
10) The cells are suspended in 1 mL sterile ultra-pure water, and then centrifuged at 2500 g for 30 s to remove the supernatant.
11) The cells are suspended in 50-100 L sterile ultra-pure water and evenly coated on the corresponding solid medium. The cells are cultivated at 30'C(sometimes at lower temperature) for 24-72 h.

1. Organize homogenate
(1) Take out the mortar after high temperature and pressure disinfection, cut 50-100mg of frozen tissue into the mortar, pour in liquid nitrogen, and grind it.
(2) add 1ml Trizol to every 50-100mg homogenized tissue sample. The quantity of sample should not exceed 10% of the Trizol volume, otherwise DNA contamination will occur.
(3) The homogenate specimens are transferred to a 1.5mL EP tube and placed at 15-30 °C for 5 minutes to completely isolate the nuclear-protein complex.
2. Phase separation
Add 0.2ml chloroform and shake it violently by hand for 15 seconds, place it at 15-30 °C for 2-3 minutes, then centrifuge it at 12,000 g, 15 min, 2-8 °C. After centrifugation, it is divided into three layers, one red layer, the phenol-chloroform phase, one intermediate layer, and one colorless layer, the aqueous phase. RNA only exists in the aqueous phase. Aqueous phase accounts for 60% of the total Trizol.
3. RNA sediment
Transfer the upper water phase to another clean EP tube, add 0.5mL isopropyl alcohol, stand for 10 minutes, 15-30 °C, then centrifuge at 12,000 g for 10 minutes, 2-8 °C. Before centrifugation, flocculent colloidal precipitation can be seen on the side wall and bottom of the tube, which is the RNA precipitation.
4. RNA washing
Remove the supernatant, add 1ml 75% ethanol to wash the RNA precipitation, mix with vortex , centrifuge at 7,500 g, 2-8 °C for 5 minutes.
5. RNA redissolution
Remove the supernatant, leave in vacuum or air for 5-10 minutes, and dry the RNA precipitation. Note that the RNA precipitate should not be completely dried, as this will greatly reduce its solubility. The A260/280 ratio of partially dissolved RNA samples was < 1.6. RNA precipitation is resuspended in RNase free water or 0.5% SDS solution. The RNA precipitation is repeatedly blown several times with the pipettor and placed at 55-60°C for 10 minutes. Store at -20°C after concentration measurement.
6. Concentration measurement:
2 μL of stored solution is added to another EP tube, and 98 μL of RNase free water is added. The od260/280 value is measured after centrifugation and mixing. 1 OD = 40 μg RNA. A value of OD260/280 between 1.8-2.0 is considered highly pure. The concentration of 1000ng/ml is more accurate. The concentration is too high to be measured after dilution.
7. RNA identification: Formaldehyde denatured agarose electrophoresis, to determine the integrity of the extracted RNA and DNA contamination.
1. Yeast is inoculated to 30 mL liquid YEPD medium and cultured at 30 ℃ for 12 h.
2. The culture is transferred to 30 mL fresh YEPD liquid medium with 1% inoculum, and cultured at 30 ℃ for 8 h, so that the cells could achieve synchronous growth state.
3. The yeast culture is placed on ice for 30 min and centrifuged at 6000 r/min for 5 min to collect the cells.
4. Discard the supernatant and add 15 mL pre-cooled ultra-pure water to wash the cells once.
5. The cells are collected by centrifugation at 6,000r /min for 5 min and washed with 15 mL 1 mol/L sorbitol for three times.
6. The cells are collected by centrifugation, add 200 μL 1 mol/L sorbitol heavy suspension cells and 200 μL suspension was taken to 1.5 mL centrifugation tube.
7. 20 μL(≤5 μg) plasmid or DNA fragment to be transformed is added into the prepared suspension, which is gently mixed and then chilled for 10 min.
8. Transfer the cell suspension after ice bath into a pre-cooled electrocuter and shock it with 1,500 V for 5 ms;
9. Add 1 mol/L sorbitol to wash the cell suspension from the electrocuter, and 200 μL of the cell suspension is coated in the medium with corresponding antibiotic.
10. Transformants are selected after 30℃ culture for 3-5 days.
(1)First,incubate yeast with foregin genes with 2% glucose in FunGenome SD /-Ura medium (YGM003A-3) at 30℃ , shaking culture at 280 rpm until its OD600 reaches 1.2-1.4 .
(2)Centrifuge the yeast culture at 1000 rpm for 5 minutes, and then discard the supernatant.
(3)Suspend the cells with 200-300ml FunGenome Sc-Ura liquid medium without any sugar; mix them well,and then centrifuge the solution and discard the supernatant.
(4) Add 500ml (Sc-U) medium without any sugar, and continue shaking for 3 hours to deplete the glucose in the cell. (Note:glucose is a strong inhibitor of the GAL promoter. In the presence of a trace amount of glucose, genes driven by the GAL1 promoter will not be expressed)
(5)Centrifuge the solution and discard the supernatant, which is to be replaced by 500ml fresh Sc-Ura medium YGM003A-3; add raffinose and galactose (the final concentration should be 2%) and continue shaking the yeast culture for 8-12 hours. If the heterologously expressed protein is toxic to yeast cells, the expression time should not exceed 5 hours. Special attention should be paid that raffinose and galactose cannot be autoclaved. It is recommended to prepare a 40% sterile mother liquor that has been filtered and sterilized
(1)Sample preparation: apply 2-3 μl of bacterial liquid after induction on a piece of clean sterile slide, and then add 3-5 μl sterile ddH2O to dilute and expand the area of bacterial liquid. Draw a circle around the bacterial solution at a diameter that is 1-2 mm longger than the one of the solution. Wait until it dires naturally.
(2)Sealing: drop Cool Polyoxymethylene (PFA) at appropriate amount onto the slide to cover the mycoderm and wait for 5 min to fix the cell morphology, and the wait until it dries naturally. (CAUTION : the mycoderm should be kept in PFA during the 5 min's waiting).
(3)Cleaning: add 200 μl 1xPBS to the mycoderm, and remove it after slight vibration.
(4)Primary antibody incubation: put the slide into a slide box with water, and then add 100 μl primary antibody( His-tag antibody,Mouse) at working concentration. Place the slide in a 37 ℃ incubator for 45 min-1h.
(5)Cleaning: remove the primary antibody with 200 μl 1xPBS. Remove 1xPBS after slight vibration. Repeat this procedure for 3 times.
(6) Secondary antibody incubation: put the slide into an opaque glass box with water, and cover the mycoderm with 100 μl secondary antibody at working concentration. Place the slide box in a 37℃ incubator for 45min-1h.
(7)Cleaning: add 200 μl 1xPBS to the mycoderm, and remove it after slight vibration.
(8)Mounting: add 1-2 drops of Antifade Mounting Medium (or glycerin) on the mycoderm ,cover it with acover glass and remove all air bubbles.
(9)Observation: observe the slide under a flourescent microscope in the dark.