Difference between revisions of "Team:Toulouse/Experiments"
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| + | <h3>Standardization of varroas and sampling</h3> | ||
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| + | <li>Bees in box with aeration and glucose </li> | ||
| + | <li>Varroas from protocol “Sampling varroas”</li> | ||
| + | <li>Gas cylinder of CO<SUB>2</SUB></li> | ||
| + | <li>Small brush </li> | ||
| + | <li>Tweezers</li> | ||
| + | <li>Petri dishes <br> Ø x h = 35 x 15 mm</li> | ||
| + | </div> | ||
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| + | <div class="subtitle" style="margin-bottom:0px;"> | ||
| + | <h3>Methods</h3> | ||
| + | </div> | ||
| + | <ol align="justify" style="font-size:15px;"> | ||
| + | <li>With small brush take varroas from Petri dish and put them on bees in box through aeration holes</li> | ||
| + | <li>Place the box in a 35°C incubator overnight. Make sure you have a bowl with water in order to have enough humidity in incubator</li> | ||
| + | <li>Take the box out of incubator</li> | ||
| + | <li>Add CO<SUB>2</SUB> from gas cylinder into the box until all bees fall down</li> | ||
| + | <li>Open the box, take a bee with tweezer and look for varroas</li> | ||
| + | <li>When you find a varroa take him with small brush and replace bee in the box</li> | ||
| + | <li>Start again step 5 and 6 until you have enough varroas</li> | ||
| + | </ol> | ||
| + | </div> | ||
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Revision as of 10:15, 2 September 2015
Experiments & Protocols
Protocols for varroa tests
Sampling of varroa
Materials
- Bee hive
- Beekeeper suit
- Gloves
- Smoker
- Dry twigs
- Tweezers
- Big brush
- Small brush
- Petri dishes
Ø x h = 35 x 15 mm
Methods
- Slip beekeeper suit and gloves on and go to bee hive
- Fire dry twigs in smoker
- Open bee hive and activate smoker to get bees inside the hive
- Take a frame out the hive and remove bees with big brush and smoker
- Close bee hive
- In the lab, put the frame on a table against the wall
- With tweezer drill hole into one beehive cell
- Remove larvae and look for varroas on larvae and on beehive cell
- If there are varroas, take them with a small brush and put them on Petri dishes
- Make sure there are two or three larvae on Petri dishes in order to allow survival of varroas
- Start again step 7 to 9 until you have enough varroas
Standardization of varroas and sampling
Materials
- Bees in box with aeration and glucose
- Varroas from protocol “Sampling varroas”
- Gas cylinder of CO2
- Small brush
- Tweezers
- Petri dishes
Ø x h = 35 x 15 mm
Methods
- With small brush take varroas from Petri dish and put them on bees in box through aeration holes
- Place the box in a 35°C incubator overnight. Make sure you have a bowl with water in order to have enough humidity in incubator
- Take the box out of incubator
- Add CO2 from gas cylinder into the box until all bees fall down
- Open the box, take a bee with tweezer and look for varroas
- When you find a varroa take him with small brush and replace bee in the box
- Start again step 5 and 6 until you have enough varroas
$$ P\cdot V = n\cdot R\cdot T, \textrm{ideal gaz law} $$ $$ P_A = C_A\cdot R\cdot T = 7,826\cdot10^{-3}\times8,314\times293=19,964 Pa $$
- PA: partial pressure of A in Pa
- CA: Concentration of A in air in mol.m-3
- R: perfect gaz constant = 8,314 J.mol-1.K-1
- T: temperature in °K
$$ P_A = H_A\cdot C_{A,eq}, \textrm{Henry's law} $$ $$ C_{A,eq} = \frac{19,964}{0,019} = 1,019mol.L^{-1}$$
- CA,eq: equivalent concentration in liquid in mol.L-1
- HA: Henry's constant = 0,019 Pa.m3mol-1
Transformation Protocol: RbCl method
Media and solution
| YETM 500mL | TFB1 200mL | TFB2 200mL |
|---|---|---|
|
|
|
Preparation of Competent Cells
- 1. Streak cells froms frozen stock onto YETM plate. Incubate overnight at 37°C
- 2. Pick a single fresh colony to inoculate 5mL of YETM medium. Grow over night at 37°C.
- Do not vortex cells at any time after this point in the procedure
- 3. Dilute 1mL of culture into 50mL YETM medium prewarmed to 37°C
- Grow at 37°C for 2hr with agitation
- Volumes can be scaled up 5X and all of the 5mL overnight culture can be used
- 4. Transfer culture to sterile 50 mL tube. Chill on ice/water 10-15 minutes
- 5. Centrifuge for 10 minutes at 2000rpm at 4°C. Immediately aspirate off all of the supernatant
- Do not allow cells to warm above 4°C at any time in this procedure
- 6. Resuspend cells in 10mL of ice-cold TFB1 with gentle re-pipetting. Use chilled glass or plastic pipette
- 7. Incubate cells on ice for 5min
- 8. Repeat step 5
- 9. Resuspend cells in 2mL of ice-cold TFB2 with gentle re-pipetting. Use micropipet tip (plastic)
- 10. Incubate cells on ice for 15 minutes
- Cells may be used for transformation or frozen
- To freeze: aliquot cell in 200μL volumes into prechilled 0.5mL microfuge tube (on ice)
- Freeze immediately in liquid nitrogen
- Store cells frozen at -80°C
Transformation of Competent Cells
- 1. If starting with frozen competent cells, warm tube/cells by gently twirling between your fingers until just thawed.
Immedately place on ice for about 5 minutes - 2. Add to 1,5mL eppendorff on ice: 2-3μL iGEM plate or 1μL plasmid or 10μL ligation.
- 3. Add 100μL of competent cells and mix by gentle re-pipetting
- 4. Incubate cells on ice for 20-30 minutes
- 5. Heat shock the cells exactly 90 seconds at 42°C
- 6. Return cells on ice for 2 minutes
- 7. Add 1mL of YETM medium. Incubate at 37°C for 45-60 minutes with slow gentle shaking
- 8. Plate 0.1-0.2 mL of transformed cells on LB-plate containing the appropriate antibiotic. Incubate overnight at 37°C
Minipreps
- 1. Resuspend 4 to 12 colonies from the plate and name each colony taken on the tubes and on the plate (A, B, C, …)
- 2. Resuspend one colony per culture tube in 5 mL of LB medium with antibiotic
- 3. Let the culture grow overnight at 37°C in a shaking incubator
- 4. Use the QIAprep spin Miniprep Kit for each culture tube. The last step consisting in the elution of the DNA is made with elution buffer or water at 55°C
- 5. Keep the tubes at -20°C
Cloning
First step: Digestion
Both parts have the same antibiotic resistance
| Vector | Insert |
|---|---|
1 µg of |
10 µL of miniprep plasmid |
1 µL of each restriction enzymes |
1 µL of each restriction enzymes |
2 µL of Green Buffer |
2 µL of Green Buffer |
9 µL of Milli-Q water |
4 µL of Milli-Q water |
Incubate 15 minutes at 37°C |
|
The two parts have a different antibiotic resistance
| Both parts |
|---|
5 µL of miniprep plasmid |
1 µL of each restriction enzymes |
2 µL of Green Buffer |
9 µL of Milli-Q water |
Incubate 15 minutes at 37°C |
Migration and gel extraction
- 1. Prepare a 1% or 2% electrophoresis agarose gel with 0.5x TAE buffer
- 2. Put 20 µL of sample + 6 µL of marker (1 kb for 1% gel and 100 pb for 2%) into the well
- 3. Migration for 30 min at 100 V or 1 hour at 50 V
- 4. Bathe 10 minutes in BET
- 5. Wash in water for 5 minutes
- 6. The gel extraction is realized thanks to the QIAGEN Gel Extraction Kit
- Two ways to inactivate the enzymes for the vector
- Use of DNA Clean up kit for the DNA fragment above 200 pb
- Heat inactivation at 95°C for 10 minutes
Second step: Ligation
| Mix | Control |
|---|---|
10 µL of insert |
no insert |
4 µL of vector |
4 µL of vector |
2 µL of 10x T4 buffer |
2 µL of 10x T4 buffer |
0.5 µL of T4 ligase |
0.5 µL of T4 ligase |
3.5 µL of Milli-Q water |
13.5 µL of Milli-Q water |
Incubate the ligation mix 15 minutes at room temperature (22°C) |
|
Keep the tubes in ice or at -20°C to prepare the transformation |
|
Third step: Ligation
- 1. Take 10µL of the ligation mix for 100 µL of competent cells and use the Toulouse iGEM Team 2015 transformation protocol
- 2. Plate the solution on selective medium overnight at 37°C
References
