Team:Evry/Protocols

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<h1>PCR with Phusion polymerase<h1>
+
<h1>Preparation of competent bacteria</h1>
 +
<i> A preculture of TOP10 cells grown in LB medium is provided</i><br>
 +
- Reseed 200ml of LB medium with 200µl of pre-culture. Incubate at 37°C (150rpm) until a DO600nm between 0,30 to 0,35 (exponential growth phase)<br>
 +
To do this, follow the evolution of DO600nm by making sterile samples of 1ml suspension. Take a first step after 1:30 of culture (generation time of bacteria is about 30min), then every half hour, using the same spectrophotometer for all measurements.<br>
 +
<b>The cells must be kept on ice for the duration of the protocol</b><br>
 +
- Transfert the cells in four Falcon tubes previously cooled in ice.<br>
 +
- Incubate the culture 30min on ice.<br>
 +
- Centrifuge at 3000rpm for 5min at 4°C.<br>
 +
- Repeat all four base in 10ml of cold 0,1M CaCl2.<br>
 +
- Centrifuge at 3000rpm for 5min at 4°C.<br>
 +
- Resuspend the pellet in 10ml of 0,1M MgCl2 solution and incubate 30min on ice.<br>
 +
- Spin at 3000rpm for 5min at 4°C and add 2ml [CaCl2 + 10% glycerol].<br>
 +
- Aliquote cells in 1,5ml Eppendorf tubes previously cooled.<br>
 +
<b> Store them on ice</b><br>
 +
<h1> Transformation </h1>
 +
<i> => With chemically lab-made competent cells</i><br>
 +
- '''Keep constantly the cells on ice'''.<br>
 +
- Add 2-10µl of DNA ligation (2µl) or miniprep (2µl) in 100ul of competent bacteria (1µl of the positive control puc19).<br>
 +
- Incubate at least 30' on ice.<br>
 +
- Heat-shock the cells during 30'' at 42°C in a water bath without shaking.<br>
 +
- Put cells for 2' on ice.<br>
 +
- Complete to 1 ml with pre-warmed SOC medium (42°C).<br>
 +
- Incubate 1h at 37°C under shaking (225 rpm).<br>
 +
- Take 100 µl and spread on appropriate plates.<br>
 +
- Incubate O/N at 37°C.<br>
-
<h1>Préparation of LB medium and LB Agar:</h1>
+
<h1>Midi-prep</h1>
 +
<i>Protocol from QIAGEN plasmid purification handbook</i><br><br>
 +
Pre-chill Buffer P3 at 4°C <br><br>
 +
<b>1.Pick a single colony</b> from a freshly streaked selective plate and inoculate a starter
 +
culture of 2–5 ml LB medium containing the appropriate selective antibiotic. Incubate
 +
for approx. 8 h at 37°C with vigorous shaking (approx. 300 rpm).<br>
 +
Use a tube or flask with a volume of at least 4 times the volume of the culture.<br><br>
 +
<b>2.Dilute the starter culture</b> 1/500 to 1/1000 into selective LB medium. For high-copy
 +
plasmids, inoculate 25 ml medium with 25–50 μl of starter culture. For low-copy plasmids, inoculate 100 ml medium with 100–200 μl of starter culture. Grow at 37°C for 12–16 h with vigorous shaking (approx. 300 rpm).<br>
 +
Use a flask or vessel with a volume of at least 4 times the volume of the culture. The
 +
culture should reach a cell density of approximately 3–4 x 109 cells per milliliter,
 +
which typically corresponds to a pellet wet weight of approximately 3 g/liter
 +
medium.<br><br>
 +
<b>3. Harvest the bacterial cells</b> by centrifugation at 6000 x g (6900rpm with rotor AV10/Ser N.0106/04/Cat N.11175754 - Centri CR31 Thermo) for 15 min at 4°C.<br>
 +
If you wish to stop the protocol and continue later, freeze the cell pellets at –20°C.<br><br>
 +
<b>4. Resuspend the bacterial pellet in 4 ml Buffer P1.</b><br>
 +
For efficient lysis it is important to use a vessel that is large enough to allow complete
 +
mixing of the lysis buffers. Ensure that RNase A has been added to Buffer P1.If LyseBlue reagent has been added to Buffer P1, vigorously shake the buffer bottle before use to ensure LyseBlue particles are completely resuspended. The bacteria should be resuspended completely by vortexing or pipetting up and down until no
 +
cell clumps remain.<br><br>
 +
<b>5. Add 4 ml Buffer P2</b>, mix thoroughly by vigorously inverting the sealedtube 4–6 times, and incubate at room temperature (15–25°C) for 5 min.<br>
 +
Do not vortex, as this will result in shearing of genomic DNA. The lysate should
 +
appear viscous. Do not allow the lysis reaction to proceed for more than 5 min. After
 +
use, the bottle containing Buffer P2 should be closed immediately to avoid
 +
acidification from CO2 in the air.<br>
 +
If LyseBlue has been added to Buffer P1 the cell suspension will turn blue after
 +
addition of Buffer P2. Mixing should result in a homogeneously colored suspension.<br>
 +
If the suspension contains localized colorless regions or if brownish cell clumps are
 +
still visible, continue mixing the solution until a homogeneously colored suspension
 +
is achieved.<br><br>
 +
<b>6. Add 4 ml of chilled Buffer P3</b>, mix immediately and thoroughly by
 +
vigorously inverting 4–6 times, and incubate on ice for 15 min.<br>
 +
Precipitation is enhanced by using chilled Buffer P3 and incubating on ice. After
 +
addition of Buffer P3, a fluffy white material forms and the lysate becomes less
 +
viscous. The precipitated material contains genomic DNA, proteins, cell debris, and
 +
KDS. The lysate should be mixed thoroughly to ensure even potassium dodecyl sulfate
 +
precipitation. If the mixture still appears viscous, more mixing is required to
 +
completely neutralize the solution.<br>
 +
If LyseBlue reagent has been used, the suspension should be mixed until all trace of
 +
blue has gone and the suspension is colorless. A homogeneous colorless suspension
 +
indicates that the SDS has been effectively precipitated.<br><br>
 +
<b>7. Centrifuge</b> at ≥20,000 x g (10000rpm with rotor AV10/Ser N.0106/04/Cat N.11175754 - Centri CR31 Thermo)for 30 min at 4°C. Remove supernatant containing plasmid
 +
DNA promptly.<br>
 +
Before loading the centrifuge, the sample should be mixed again. Centrifugation
 +
should be performed in non-glass tubes (e.g., polypropylene). After centrifugation
 +
the supernatant should be clear.<br>
 +
Note: Instead of centrifugation steps 7 and 8, the lysate can be efficiently cleared by
 +
filtration using a QIAfilter Kits or Cartridges (see www.qiagen.com/products/
 +
plasmid/LargeScaleKits).<br><br>
 +
<b>8. Centrifuge the supernatant again</b> at ≥20,000 x g (10000rpm with rotor AV10/Ser N.0106/04/Cat N.11175754 - Centri CR31 Thermo) for 15 min at 4°C. Remove
 +
supernatant containing plasmid DNA promptly.<br>
 +
This second centrifugation step should be carried out to avoid applying suspended
 +
or particulate material to the QIAGEN-tip. Suspended material (causing the sample
 +
to appear turbid) can clog the QIAGEN-tip and reduce or eliminate gravity flow.<br><br>
 +
<b> 9. Equilibrate a QIAGEN-tip 100 </b>by applying 4 ml Buffer QBT, and allow the column to empty by gravity flow.<br>
 +
Flow of buffer will begin automatically by reduction in surface tension due to the
 +
presence of detergent in the equilibration buffer. Allow the QIAGEN-tip to drain
 +
completely. QIAGEN-tips can be left unattended, since the flow of buffer will stop
 +
when the meniscus reaches the upper frit in the column.<br><br>
 +
<b>10. Apply the supernatant from step 8 to the QIAGEN-tip</b> and allow it to enter the resin
 +
by gravity flow.<br>
 +
The supernatant should be loaded onto the QIAGEN-tip promptly. If it is left too long
 +
and becomes cloudy due to further precipitation of protein, it must be centrifuged
 +
again or filtered before loading to prevent clogging of the QIAGEN-tip.<br><br>
 +
<b>11. Wash the QIAGEN-tip </b>with 2 x 10 ml Buffer QC.<br>
 +
Allow Buffer QC to move through the QIAGEN-tip by gravity flow. The first wash is
 +
sufficient to remove all contaminants in the majority of plasmid DNA preparations.
 +
The second wash is especially necessary when large culture volumes or bacterial
 +
strains producing large amounts of carbohydrates are used.<br><br>
 +
<b>12. Elute DNA with 5 ml Buffer QF.</b><br>
 +
Collect the eluate in a 15 ml or 50 ml tube (not supplied). Use of polycarbonate
 +
centrifuge tubes is not recommended as polycarbonate is not resistant to the alcohol
 +
used in subsequent steps.<br><br>
 +
<b>13. Precipitate DNA</b> by adding 3.5 ml (0.7 volume) room-temperature
 +
isopropanol to the eluted DNA. Mix and centrifuge immediately at ≥15,000 x g for
 +
30 min at 4°C. Carefully decant the supernatant.<br>
 +
All solutions should be at room temperature in order to minimize salt precipitation,
 +
although centrifugation is carried out at 4°C to prevent overheating of the sample.<br>
 +
Alternatively, disposable conical bottom centrifuge tubes can be used for
 +
centrifugation at 5000 x g (10000rpm with rotor AV10/Ser N.0106/04/Cat N.11175754 - Centri CR31 Thermo)for 60 min at 4°C. Isopropanol pellets have a glassy
 +
appearance and may be more difficult to see than the fluffy, salt-containing pellets
 +
that result from ethanol precipitation. Marking the outside of the tube before
 +
centrifugation allows the pellet to be more easily located. Isopropanol pellets are also
 +
more loosely attached to the side of the tube, and care should be taken when
 +
removing the supernatant.<br><br>
 +
<b>14. Wash DNA pellet</b> with 2 ml of room-temperature 70% ethanol, and
 +
centrifuge at ≥15,000 x g (10000rpm with rotor AV10/Ser N.0106/04/Cat N.11175754 - Centri CR31 Thermo)for 10 min. Carefully decant the supernatant without
 +
disturbing the pellet.<br>
 +
The 70% ethanol removes precipitated
 +
salt and replaces isopropanol with the more volatile ethanol, making the DNA easier
 +
to redissolve.<br><br>
 +
<b>15. Air-dry the pellet</b> for 5–10 min, and redissolve the DNA in a suitable volume of buffer
 +
(e.g., TE buffer, pH 8.0, or 10 mM Tris·Cl, pH 8.5).<br>
 +
Redissolve the DNA pellet by rinsing the walls to recover all the DNA, especially if
 +
glass tubes have been used. Pipetting the DNA up and down to promote
 +
resuspension may cause shearing and should be avoided. Overdrying the pellet will
 +
make the DNA difficult to redissolve. DNA dissolves best under slightly alkaline
 +
conditions; it does not easily dissolve in acidic buffers.<br><br>
 +
 
 +
<h1>DNA purification phenol/chloroforme</h1>
 +
Add 1 volume of phenol:chlorophorme:isoamyalcool (25:24:1)<br>
 +
Vortex<br>
 +
Centrifuge 10min, 14000rpm, 4°C<br>
 +
Recovery the aqueous phase (sup) and transfert it in a new tube<br>
 +
Add 1 volume pf chloroforme:isoamylalcool (24:1)<br>
 +
Vortex<br>
 +
Centrifuge 10min, 14000rpm, 4°C<br>
 +
Recovery the aqueous phase (sup) and transfert it in a new tube<br>
 +
Precipitate by adding 0,7 volume of isopropanol and 0,3 volume of aminium acetate 7,5M<br>
 +
Mix by inversion and stock at -80°C for 30min or at -20°C over night<br>
 +
Centrifuge 30min, 14000rpm, 4°C<br>
 +
Eliminate the supernatant and wash the pellet with 500ul EtOH 70%<br>
 +
Centrifuge 10min, 14000rpm, 4°C<br>
 +
Eliminate EtOH and dry for 10min at room temperature<br>
 +
Resuspend the pellet in TE buffer 1X<br>
 +
 
 +
 
 +
 
 +
<h1>PCR with Phusion High-Fidelity DNA Polymerase</h1>
 +
 
 +
<h2>Tube preparation</h2>
 +
Put items in this order:
 +
 
 +
<table border>
 +
      <tr>
 +
            <td>Component</td>
 +
            <td>50µl reaction</td>
 +
            <td>Comments</td>
 +
      </tr>
 +
      <tr>
 +
            <td>H2O</td>
 +
            <td>32</td>
 +
            <td></td>
 +
      </tr>
 +
      <tr>
 +
            <td>5x Phusion HF Buffer</td>
 +
            <td>10</td>
 +
            <td></td>
 +
      </tr>
 +
      <tr>
 +
            <td>10mM dNTPs</td>
 +
            <td>1</td>
 +
            <td></td>
 +
      </tr>
 +
      <tr>
 +
            <td>Primer FW</td>
 +
            <td>2</td>
 +
            <td>Primers have to be at 10µM</td>
 +
      </tr>
 +
      <tr>
 +
            <td>Primer RV</td>
 +
            <td>2</td>
 +
            <td>Primers have to be at 10µM</td>
 +
      </tr>
 +
      <tr>
 +
            <td>Template DNA</td>
 +
            <td>1</td>
 +
            <td></td>
 +
      </tr>
 +
      <tr>
 +
            <td>DMSO (optional)</td>
 +
            <td>1,5</td>
 +
            <td>recommended for GC-rich amplicons < 20kb</td>
 +
      </tr>
 +
      <tr>
 +
            <td>Phusion DNA polymerase</td>
 +
            <td>0,5</td>
 +
            <td></td>
 +
      </tr>
 +
</table>
 +
 
 +
<h2>Cycling instructions</h2>
 +
 
 +
<table border>
 +
      <tr>
 +
          <td>Cycle step</td>
 +
          <td>Temperature</td>
 +
          <td>Time</td>
 +
          <td>Cycles</td>
 +
      </tr>
 +
      <tr>
 +
          <td>Initial denaturation</td>
 +
          <td>98°C</td>
 +
          <td>4min</td>
 +
          <td>1</td>
 +
      </tr>
 +
      <tr>
 +
          <td>Denaturation</td>
 +
          <td>98°C</td>
 +
          <td>20s</td>
 +
          <td rowspan="3">30</td>
 +
      </tr>
 +
      <tr>
 +
          <td>Annealing</td>
 +
          <td>Lower Tm of primers</td>
 +
          <td>30s</td>
 +
      </tr>
 +
      <tr>
 +
          <td>Extension</td>
 +
          <td>72°C</td>
 +
          <td>30S/kb</td>
 +
      </tr>
 +
      <tr>
 +
          <td rowspan="2">Final extension</td>
 +
          <td>72°C</td>
 +
          <td>10min</td>
 +
          <td rowspan="2">1</td>
 +
      </tr>
 +
      <tr>
 +
          <td>4°C</td>
 +
          <td>hold</td>
 +
      </tr>
 +
</table>
 +
 
 +
<h1>Preparation of LB medium and LB Agar:</h1>
Line 16: Line 253:
Autoclaved at 250°C
Autoclaved at 250°C
 +
 +
<h1>Gel extraction</h1>
 +
1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel under UV light. <br>
 +
2. Weight the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel. <br>
 +
3. Incubate at 50°C for 10 min, until the gel slice has completely dissolved. <br>
 +
4. The color of the mixture have to be yellow, otherwise add 10 µl of 3M sodium acetate. <br>
 +
5. Ass 1 gel volume of isopropanol to the sample and mix. <br>
 +
6. Place a spin column in a provided 2 ml collection tube. <br>
 +
7. To bind DNA, apply the sample to the column and centrifugat for 1 min. Discard the flow-through and place the column back into the same tube.<br>
 +
8. To wash, add 0,75 ml of buffer PE to the column and centrifugate for 1 min.Discard the flow-through and place the column back into the same tube.<br>
 +
9. Centrifugate the column in a 2 ml Collection tube for 1 min 17,900xg (13,000 rpm).<br>
 +
10. Place the column into a clean 1,5 ml microcentrifuge tube.<br>
 +
11. To elute DNA, add 15 µl of water to the column and centrifugate for 1 min.<br>
----
----
 +
 +
<h1>In vitro fertilization of <i>Xenopus tropicalis</i></h1>
 +
 +
<h3>2 or 3 days before the IVF</h3>
 +
<b>Pre-injection of frogs (males et females)</b><br>
 +
100µl of hCG (0,1UI/µl, do a dilution by 10 of the commercial solution)<br>
 +
 +
<h3>D day</h3>
 +
 +
Test the injector before beginning<br>
 +
Inject the 100µl hCG (1UI/µl) in the morning<br>
 +
Isolate the animals : a female by box<br>
 +
 +
<b>Prepare :</b><br>
 +
Timer <br>
 +
Methylcellulose (put at room temperature)<br>
 +
Boxes special for injections<br>
 +
Materiel of injection <br>
 +
Capillaires <i>ad hoc</i> with oil<br>
 +
Solutions to inject (add 1µl of blue or red of Nile)<br>
 +
Solution L15/FCS : <br>
 +
- Petri dishes (1/femelle, d=10 cm) lined of L15/FCS (500µl)<br>
 +
- 2 tubes 1,5mL with 250µl of L15/FCS  (one for each testicle)<br>
 +
Pestles in plastic cleaned to grind the testicles<br>
 +
MMR 0,1X<br>
 +
MMR 0,05X<br>
 +
MS-222 0,2% <br>
 +
 +
<h2>Euthanasia of the male</h2>
 +
[[File:Photo_Dissection.jpeg|left|100px]]
 +
Put the male in a box with MS-222 0,2% and wait until he doesn't response for pinches. Take the male out and process the dissection to extract the two testicles.<br>
 +
For that, dispose the animal: the dorsal face against the dissection board. Pinch the skin in the middle of the belly and do a small incision with scissors; from this point, perform skin incisions shown in the diagram.<br><br><br>
 +
[[File:Photo_Testicles.jpeg|left|100px]]
 +
Fold the flaps and skin incision in the muscular wall avoiding severing the abdominal vein to avoid hemorrhage. Both gonads are located in the ventral face of kidneys. <br>
 +
Both gonads are located in the ventral surface of the kidneys, they cover more or less and in <i>corpora lutea</i> (fat mass).By extracting them from the abdominal cavity, we can extract the testicles attached to them. The male's testicles are two oval organs, smooth, yellow-pale as can be seen in the photograph below.<br>
 +
Roll the testicles on kitchen paper to remove blood and blood vessels. Dip each testicle in a tube of 1,5mL with 250µl of L15/FCS. Keep tubes on ice. Crush the testicles with a pestle until obtaining a homogeneous solution. Add 250µl of L15/FCS, mix with a P1000 always on ice.<br>
 +
 +
<h2>Lay eggs of females</h2>
 +
Spawn females (who have already started laying alone) in Petri dishes previously coated with L15/FCS (gently massage into their flanks).<br>
 +
 +
<h2>In vitro fertilization</h2>
 +
With a pasteur pipette in plastic, remove as much liquid in the Petri dish prior the IVF. Add 50µl/500eggs of sperm solution on the eggs with P1000 and mix with a pipette tip to distribute the sperm on the eggs. Let the sperm fix on the eggs during 3-5 min.<br>
 +
Activate the spermatozoid by recovering the eggs of MMR 0,05X to allow the fecundation.<br>
 +
 +
<h2>Dejellying embryos</h2>
 +
15 min after fertilization activation, processing of the degangage.<br>
 +
For that, prepare cysteine 2%, pH 7,5-8 :<br>
 +
- Weight 2g of cysteine powder, suspend it in 80 mL of MMR 0,1% and put it in a beaker under magnetic agitation. Mesure pH and adjust it by adding NaOH 1M. <br>
 +
- Complete with MMR 0,1% to 100mL.<br>
 +
Remove MMR 0,05% from the Petri dish containing the eggs. Add the cysteine and agitate the plate by rotation for 2 min.<br> Transfer eggs to a 100ml pot (red plug) and continue to agitate by rotation for 5-8 min. Make sure that the embryos seem to touch each other to validate the degangage.<br>
 +
Rinse 3 times with MMR 0,1X.<br>
 +
 +
Attention : during the diferent phases of washing, ensure that the eggs are always in liquid.<br>
 +
 +
Add some methylcellulose to eggs to delay the developpement and facilitate the injection. <br>
 +
 +
Place 50-100 eggs in a special injection Petri dish (lined with a small mesh) and inject DNA.<br>
 +
 +
After injections, the embryos are transfered to MMR 0,1x in small petri dishes lined in the background with agarose. Boxes are incubated at 21°C. <br>
 +
 +
<h1>Auxin toxicity test</h1>
 +
<b>Aim :</b> test the toxicity of auxin
 +
 +
 +
<h2> Preparation of stock solutions 0,05M</h2>
 +
 +
<b>IAA (MW = 175 mol/g)</b><br>
 +
IAA powder stocked at -20°C<br>
 +
0,0876g in 10ml NaOH 0,1M <br>
 +
<b>NAA (MW = 186 mol/g)</b><br>
 +
NAA powder stocked at room temperature<br>
 +
0,0931g in 10ml NaOH 0,1M<br><br>
 +
Stocked at 4°C<br>
 +
 +
<h2>Day 1</h2>
 +
 +
<h3>Preparation of dilutions</h3>
 +
<b>Preparation of the mother solution </b><br>
 +
0,4ml IAA (or NAA) 0,05M<br>
 +
15ml MMR 0,1X<br>
 +
Ajust pH with HCl 1N<br>
 +
Fill up to the 20ml with MMR 0,1X<br>
 +
 +
<b>Solution n°1, 500uM :</b><br>
 +
7 ml mother solution + 7ml MMR 0,1X<br>
 +
 +
<b>Solution n°2, 250uM :</b><br>
 +
6ml solution n°1 + 6ml MMR 0,1X<br>
 +
 +
<b>Solution n°3, 125uM :</b><br>
 +
4ml solution n°2 + 4ml MMR 0,1X<br>
 +
 +
<h3>Filling the wells with prepared solutions (~2ml/weel</h3>
 +
 +
<table border>
 +
      <tr>
 +
          <td><i>12-wel plate</i></td>
 +
          <td>1</td>
 +
          <td>2</td>
 +
          <td>3</td>
 +
          <td>4</td>
 +
      </tr>
 +
      <tr>
 +
          <td><i>A</i></td>
 +
          <td>MMR 0,1X</td>
 +
          <td>Solution n°3</td>
 +
          <td>Solution n°2</td>
 +
          <td>Solution n°1</td>
 +
      </tr>
 +
      <tr>
 +
          <td><i>B</i></td>
 +
          <td>MMR 0,1X</td>
 +
          <td>Solution n°3</td>
 +
          <td>Solution n°2</td>
 +
          <td>Solution n°1</td>
 +
      </tr>      <tr>
 +
          <td><i>C</i></td>
 +
          <td>MMR 0,1X</td>
 +
          <td>Solution n°3</td>
 +
          <td>Solution n°2</td>
 +
          <td>Solution n°1</td>
 +
      </tr>
 +
</table><br>
 +
 +
<h3> Add the embryos (see IVF protocol)</h3>
 +
Add 10-30 embryos/well
 +
 +
<h3> Incubation </h3>
 +
Incubator 21°C
 +
 +
<h2> Days after </h2>
 +
 +
<h3>Microscope observation</h3>
 +
Take a picture of each well where it’s possible to see all the embryos<br>
 +
Count the alive and dead embryos<br>
 +
Remove the dead embryos<br>
 +
 +
<h3>Medium changing</h3>
 +
Protocol of DAY 1 for the preparation<br>
 +
Incline the plate and pipet the medium with a Pasteur pipette<br>
 +
Add MMR 0,1X in each well<br>
 +
Incline the plate and pipet the medium with a Pasteur pipette<br>
 +
Add the new medium following this map:<br>
 +
 +
 +
<table border>
 +
      <tr>
 +
          <td><i>12-wel plate</i></td>
 +
          <td>1</td>
 +
          <td>2</td>
 +
          <td>3</td>
 +
          <td>4</td>
 +
      </tr>
 +
      <tr>
 +
          <td><i>A</i></td>
 +
          <td>MMR 0,1X</td>
 +
          <td>Solution n°3</td>
 +
          <td>Solution n°2</td>
 +
          <td>Solution n°1</td>
 +
      </tr>
 +
      <tr>
 +
          <td><i>A</i></td>
 +
          <td>MMR 0,1X</td>
 +
          <td>Solution n°3</td>
 +
          <td>Solution n°2</td>
 +
          <td>Solution n°1</td>
 +
      </tr>      <tr>
 +
          <td><i>A</i></td>
 +
          <td>MMR 0,1X</td>
 +
          <td>Solution n°3</td>
 +
          <td>Solution n°2</td>
 +
          <td>Solution n°1</td>
 +
      </tr>
 +
</table>
 +
 +
<h1>Preparation of tadpole samples for HPLC</h1>
 +
<i>The original protocol from Vastag L, Jorgensen P, Peshkin L, Wei R, Rabinowitz JD, et al. (2011) Remodeling of the Metabolome during Early Frog Development. PLoS ONE 6(2): e16881. doi:10.1371/journal.pone.0016881 article was performed on 1 egg of Xenopus laevis. Because 1 embryo of this species represents 10 embryos of Xenopus tropicalis,  we decided to perform our protocol on 10 Xenopus tropicalis embryos.</i><br>
 +
 +
<b>Aim:</b> HPLC test to evaluate the presence of auxin in tadpoles embryos<br>
 +
 +
<h2>Material</h2>
 +
- IAA powder (stocked at -20°C)<br>
 +
- NAA powder (stocked at room temperature)<br>
 +
- Acetonitrile 100% HPLC grade<br>
 +
- Methanol 100% HPLC grade<br>
 +
- Falcon 15ml<br>
 +
- 1,5ml tubes<br>
 +
- Pasteur pipettes<br>
 +
- Mortar<br>
 +
- Centrifuge<br>
 +
- Ice<br>
 +
- Vortex<br>
 +
- P1000, P200 with tips<br>
 +
 +
<b>Prerequisite:</b> To have perform a FIV and the auxin toxicity protocol the day before.<br>
 +
<b>Before beginning:</b> Turn on the centrifuge at 4°C<br>
 +
 +
<h2>Preparation of solutions (under chemical hood)</h2>
 +
<b>Mix for 10 samples</b><br>
 +
2ml acetonitrile<br>
 +
2ml methanol<br>
 +
1ml H2O<br>
 +
Cool mix to -20oC<br>
 +
<b>Prepare 1mg/ml auxin solutions in 20% acetonitrile/H2O</b><br>
 +
<h2>Preparation of the samples</h2>
 +
Take 10 embryos from each concentration of IAA and NAA (0, 125, 250 and 500 µM)<br>
 +
Rinse the embryos 4 times in H2O<br>
 +
Place the embryos in 1,5ml tubes<br>
 +
Add 55µl of cold mix<br>
 +
Crush the embryos with a mortar <br>
 +
Vortex<br>
 +
Place on ice for 20min<br>
 +
Centrifuge 5min – 14000 rpm – 4°C (centrifuge eppendorf 5417R)<br>
 +
Collect as much as possible of the supernatant and place it in new tubes<br><br>
 +
<b>To extract more:</b><br>
 +
Add 55µl of the mix on the pellet<br>
 +
Repeat all steps<br>
 +
<h1>Salkowski assay</h1>
 +
<i>Salkowski assay is the simplest way to test whether our samples (bacteria or tadpoles extracts)contain auxins.</i><br>
 +
<h2>Preparation of bacteria culture for assay</h2>
 +
1. Measure the O.D. at 600nm of bacteria in order to observe difference in growth. <br>
 +
2. Centrifuge culture and filter supernatant through 0,2 µM filter. <br>
 +
<h2>Salkowski assay</h2>
 +
1. Prepare the reagent: <br>
 +
Mix 0,5mL of FeCl3 0,5M with 25mL of HClO4 35%. Store in absence of light. <br>
 +
2. Add Salkowski reagent to the filtered supernatant in a ratio 2:1.<br>
 +
3. Incubate in dark for 30min. <br>
 +
4. Measure the D.O. at 530nm. <br>

Latest revision as of 20:34, 26 October 2012

Contents

Preparation of competent bacteria

A preculture of TOP10 cells grown in LB medium is provided
- Reseed 200ml of LB medium with 200µl of pre-culture. Incubate at 37°C (150rpm) until a DO600nm between 0,30 to 0,35 (exponential growth phase)
To do this, follow the evolution of DO600nm by making sterile samples of 1ml suspension. Take a first step after 1:30 of culture (generation time of bacteria is about 30min), then every half hour, using the same spectrophotometer for all measurements.
The cells must be kept on ice for the duration of the protocol
- Transfert the cells in four Falcon tubes previously cooled in ice.
- Incubate the culture 30min on ice.
- Centrifuge at 3000rpm for 5min at 4°C.
- Repeat all four base in 10ml of cold 0,1M CaCl2.
- Centrifuge at 3000rpm for 5min at 4°C.
- Resuspend the pellet in 10ml of 0,1M MgCl2 solution and incubate 30min on ice.
- Spin at 3000rpm for 5min at 4°C and add 2ml [CaCl2 + 10% glycerol].
- Aliquote cells in 1,5ml Eppendorf tubes previously cooled.
Store them on ice

Transformation

=> With chemically lab-made competent cells
- Keep constantly the cells on ice.
- Add 2-10µl of DNA ligation (2µl) or miniprep (2µl) in 100ul of competent bacteria (1µl of the positive control puc19).
- Incubate at least 30' on ice.
- Heat-shock the cells during 30 at 42°C in a water bath without shaking.
 - Put cells for 2' on ice.
- Complete to 1 ml with pre-warmed SOC medium (42°C).
- Incubate 1h at 37°C under shaking (225 rpm).
- Take 100 µl and spread on appropriate plates.
- Incubate O/N at 37°C.

Midi-prep

Protocol from QIAGEN plasmid purification handbook

Pre-chill Buffer P3 at 4°C

1.Pick a single colony from a freshly streaked selective plate and inoculate a starter culture of 2–5 ml LB medium containing the appropriate selective antibiotic. Incubate for approx. 8 h at 37°C with vigorous shaking (approx. 300 rpm).
Use a tube or flask with a volume of at least 4 times the volume of the culture.

2.Dilute the starter culture 1/500 to 1/1000 into selective LB medium. For high-copy plasmids, inoculate 25 ml medium with 25–50 μl of starter culture. For low-copy plasmids, inoculate 100 ml medium with 100–200 μl of starter culture. Grow at 37°C for 12–16 h with vigorous shaking (approx. 300 rpm).
Use a flask or vessel with a volume of at least 4 times the volume of the culture. The culture should reach a cell density of approximately 3–4 x 109 cells per milliliter, which typically corresponds to a pellet wet weight of approximately 3 g/liter medium.

3. Harvest the bacterial cells by centrifugation at 6000 x g (6900rpm with rotor AV10/Ser N.0106/04/Cat N.11175754 - Centri CR31 Thermo) for 15 min at 4°C.
If you wish to stop the protocol and continue later, freeze the cell pellets at –20°C.

4. Resuspend the bacterial pellet in 4 ml Buffer P1.
For efficient lysis it is important to use a vessel that is large enough to allow complete mixing of the lysis buffers. Ensure that RNase A has been added to Buffer P1.If LyseBlue reagent has been added to Buffer P1, vigorously shake the buffer bottle before use to ensure LyseBlue particles are completely resuspended. The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain.

5. Add 4 ml Buffer P2, mix thoroughly by vigorously inverting the sealedtube 4–6 times, and incubate at room temperature (15–25°C) for 5 min.
Do not vortex, as this will result in shearing of genomic DNA. The lysate should appear viscous. Do not allow the lysis reaction to proceed for more than 5 min. After use, the bottle containing Buffer P2 should be closed immediately to avoid acidification from CO2 in the air.
If LyseBlue has been added to Buffer P1 the cell suspension will turn blue after addition of Buffer P2. Mixing should result in a homogeneously colored suspension.
If the suspension contains localized colorless regions or if brownish cell clumps are still visible, continue mixing the solution until a homogeneously colored suspension is achieved.

6. Add 4 ml of chilled Buffer P3, mix immediately and thoroughly by vigorously inverting 4–6 times, and incubate on ice for 15 min.
Precipitation is enhanced by using chilled Buffer P3 and incubating on ice. After addition of Buffer P3, a fluffy white material forms and the lysate becomes less viscous. The precipitated material contains genomic DNA, proteins, cell debris, and KDS. The lysate should be mixed thoroughly to ensure even potassium dodecyl sulfate precipitation. If the mixture still appears viscous, more mixing is required to completely neutralize the solution.
If LyseBlue reagent has been used, the suspension should be mixed until all trace of blue has gone and the suspension is colorless. A homogeneous colorless suspension indicates that the SDS has been effectively precipitated.

7. Centrifuge at ≥20,000 x g (10000rpm with rotor AV10/Ser N.0106/04/Cat N.11175754 - Centri CR31 Thermo)for 30 min at 4°C. Remove supernatant containing plasmid DNA promptly.
Before loading the centrifuge, the sample should be mixed again. Centrifugation should be performed in non-glass tubes (e.g., polypropylene). After centrifugation the supernatant should be clear.
Note: Instead of centrifugation steps 7 and 8, the lysate can be efficiently cleared by filtration using a QIAfilter Kits or Cartridges (see www.qiagen.com/products/ plasmid/LargeScaleKits).

8. Centrifuge the supernatant again at ≥20,000 x g (10000rpm with rotor AV10/Ser N.0106/04/Cat N.11175754 - Centri CR31 Thermo) for 15 min at 4°C. Remove supernatant containing plasmid DNA promptly.
This second centrifugation step should be carried out to avoid applying suspended or particulate material to the QIAGEN-tip. Suspended material (causing the sample to appear turbid) can clog the QIAGEN-tip and reduce or eliminate gravity flow.

9. Equilibrate a QIAGEN-tip 100 by applying 4 ml Buffer QBT, and allow the column to empty by gravity flow.
Flow of buffer will begin automatically by reduction in surface tension due to the presence of detergent in the equilibration buffer. Allow the QIAGEN-tip to drain completely. QIAGEN-tips can be left unattended, since the flow of buffer will stop when the meniscus reaches the upper frit in the column.

10. Apply the supernatant from step 8 to the QIAGEN-tip and allow it to enter the resin by gravity flow.
The supernatant should be loaded onto the QIAGEN-tip promptly. If it is left too long and becomes cloudy due to further precipitation of protein, it must be centrifuged again or filtered before loading to prevent clogging of the QIAGEN-tip.

11. Wash the QIAGEN-tip with 2 x 10 ml Buffer QC.
Allow Buffer QC to move through the QIAGEN-tip by gravity flow. The first wash is sufficient to remove all contaminants in the majority of plasmid DNA preparations. The second wash is especially necessary when large culture volumes or bacterial strains producing large amounts of carbohydrates are used.

12. Elute DNA with 5 ml Buffer QF.
Collect the eluate in a 15 ml or 50 ml tube (not supplied). Use of polycarbonate centrifuge tubes is not recommended as polycarbonate is not resistant to the alcohol used in subsequent steps.

13. Precipitate DNA by adding 3.5 ml (0.7 volume) room-temperature isopropanol to the eluted DNA. Mix and centrifuge immediately at ≥15,000 x g for 30 min at 4°C. Carefully decant the supernatant.
All solutions should be at room temperature in order to minimize salt precipitation, although centrifugation is carried out at 4°C to prevent overheating of the sample.
Alternatively, disposable conical bottom centrifuge tubes can be used for centrifugation at 5000 x g (10000rpm with rotor AV10/Ser N.0106/04/Cat N.11175754 - Centri CR31 Thermo)for 60 min at 4°C. Isopropanol pellets have a glassy appearance and may be more difficult to see than the fluffy, salt-containing pellets that result from ethanol precipitation. Marking the outside of the tube before centrifugation allows the pellet to be more easily located. Isopropanol pellets are also more loosely attached to the side of the tube, and care should be taken when removing the supernatant.

14. Wash DNA pellet with 2 ml of room-temperature 70% ethanol, and centrifuge at ≥15,000 x g (10000rpm with rotor AV10/Ser N.0106/04/Cat N.11175754 - Centri CR31 Thermo)for 10 min. Carefully decant the supernatant without disturbing the pellet.
The 70% ethanol removes precipitated salt and replaces isopropanol with the more volatile ethanol, making the DNA easier to redissolve.

15. Air-dry the pellet for 5–10 min, and redissolve the DNA in a suitable volume of buffer (e.g., TE buffer, pH 8.0, or 10 mM Tris·Cl, pH 8.5).
Redissolve the DNA pellet by rinsing the walls to recover all the DNA, especially if glass tubes have been used. Pipetting the DNA up and down to promote resuspension may cause shearing and should be avoided. Overdrying the pellet will make the DNA difficult to redissolve. DNA dissolves best under slightly alkaline conditions; it does not easily dissolve in acidic buffers.

DNA purification phenol/chloroforme

Add 1 volume of phenol:chlorophorme:isoamyalcool (25:24:1)
Vortex
Centrifuge 10min, 14000rpm, 4°C
Recovery the aqueous phase (sup) and transfert it in a new tube
Add 1 volume pf chloroforme:isoamylalcool (24:1)
Vortex
Centrifuge 10min, 14000rpm, 4°C
Recovery the aqueous phase (sup) and transfert it in a new tube
Precipitate by adding 0,7 volume of isopropanol and 0,3 volume of aminium acetate 7,5M
Mix by inversion and stock at -80°C for 30min or at -20°C over night
Centrifuge 30min, 14000rpm, 4°C
Eliminate the supernatant and wash the pellet with 500ul EtOH 70%
Centrifuge 10min, 14000rpm, 4°C
Eliminate EtOH and dry for 10min at room temperature
Resuspend the pellet in TE buffer 1X


PCR with Phusion High-Fidelity DNA Polymerase

Tube preparation

Put items in this order:

Component 50µl reaction Comments
H2O 32
5x Phusion HF Buffer 10
10mM dNTPs 1
Primer FW 2 Primers have to be at 10µM
Primer RV 2 Primers have to be at 10µM
Template DNA 1
DMSO (optional) 1,5 recommended for GC-rich amplicons < 20kb
Phusion DNA polymerase 0,5

Cycling instructions

Cycle step Temperature Time Cycles
Initial denaturation 98°C 4min 1
Denaturation 98°C 20s 30
Annealing Lower Tm of primers 30s
Extension 72°C 30S/kb
Final extension 72°C 10min 1
4°C hold

Preparation of LB medium and LB Agar:


=> LB Agar : -18,5g LB Agar -300ml H2O

=> LB medium : -6g LB broth -300ml de H2O

Autoclaved at 250°C

Gel extraction

1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel under UV light.
2. Weight the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel.
3. Incubate at 50°C for 10 min, until the gel slice has completely dissolved.
4. The color of the mixture have to be yellow, otherwise add 10 µl of 3M sodium acetate.
5. Ass 1 gel volume of isopropanol to the sample and mix.
6. Place a spin column in a provided 2 ml collection tube.
7. To bind DNA, apply the sample to the column and centrifugat for 1 min. Discard the flow-through and place the column back into the same tube.
8. To wash, add 0,75 ml of buffer PE to the column and centrifugate for 1 min.Discard the flow-through and place the column back into the same tube.
9. Centrifugate the column in a 2 ml Collection tube for 1 min 17,900xg (13,000 rpm).
10. Place the column into a clean 1,5 ml microcentrifuge tube.
11. To elute DNA, add 15 µl of water to the column and centrifugate for 1 min.


In vitro fertilization of Xenopus tropicalis

2 or 3 days before the IVF

Pre-injection of frogs (males et females)
100µl of hCG (0,1UI/µl, do a dilution by 10 of the commercial solution)

D day

Test the injector before beginning
Inject the 100µl hCG (1UI/µl) in the morning
Isolate the animals : a female by box

Prepare :
Timer
Methylcellulose (put at room temperature)
Boxes special for injections
Materiel of injection
Capillaires ad hoc with oil
Solutions to inject (add 1µl of blue or red of Nile)
Solution L15/FCS :
- Petri dishes (1/femelle, d=10 cm) lined of L15/FCS (500µl)
- 2 tubes 1,5mL with 250µl of L15/FCS (one for each testicle)
Pestles in plastic cleaned to grind the testicles
MMR 0,1X
MMR 0,05X
MS-222 0,2%

Euthanasia of the male

Photo Dissection.jpeg

Put the male in a box with MS-222 0,2% and wait until he doesn't response for pinches. Take the male out and process the dissection to extract the two testicles.
For that, dispose the animal: the dorsal face against the dissection board. Pinch the skin in the middle of the belly and do a small incision with scissors; from this point, perform skin incisions shown in the diagram.


Photo Testicles.jpeg

Fold the flaps and skin incision in the muscular wall avoiding severing the abdominal vein to avoid hemorrhage. Both gonads are located in the ventral face of kidneys.
Both gonads are located in the ventral surface of the kidneys, they cover more or less and in corpora lutea (fat mass).By extracting them from the abdominal cavity, we can extract the testicles attached to them. The male's testicles are two oval organs, smooth, yellow-pale as can be seen in the photograph below.
Roll the testicles on kitchen paper to remove blood and blood vessels. Dip each testicle in a tube of 1,5mL with 250µl of L15/FCS. Keep tubes on ice. Crush the testicles with a pestle until obtaining a homogeneous solution. Add 250µl of L15/FCS, mix with a P1000 always on ice.

Lay eggs of females

Spawn females (who have already started laying alone) in Petri dishes previously coated with L15/FCS (gently massage into their flanks).

In vitro fertilization

With a pasteur pipette in plastic, remove as much liquid in the Petri dish prior the IVF. Add 50µl/500eggs of sperm solution on the eggs with P1000 and mix with a pipette tip to distribute the sperm on the eggs. Let the sperm fix on the eggs during 3-5 min.
Activate the spermatozoid by recovering the eggs of MMR 0,05X to allow the fecundation.

Dejellying embryos

15 min after fertilization activation, processing of the degangage.
For that, prepare cysteine 2%, pH 7,5-8 :
- Weight 2g of cysteine powder, suspend it in 80 mL of MMR 0,1% and put it in a beaker under magnetic agitation. Mesure pH and adjust it by adding NaOH 1M.
- Complete with MMR 0,1% to 100mL.
Remove MMR 0,05% from the Petri dish containing the eggs. Add the cysteine and agitate the plate by rotation for 2 min.
Transfer eggs to a 100ml pot (red plug) and continue to agitate by rotation for 5-8 min. Make sure that the embryos seem to touch each other to validate the degangage.
Rinse 3 times with MMR 0,1X.

Attention : during the diferent phases of washing, ensure that the eggs are always in liquid.

Add some methylcellulose to eggs to delay the developpement and facilitate the injection.

Place 50-100 eggs in a special injection Petri dish (lined with a small mesh) and inject DNA.

After injections, the embryos are transfered to MMR 0,1x in small petri dishes lined in the background with agarose. Boxes are incubated at 21°C.

Auxin toxicity test

Aim : test the toxicity of auxin


Preparation of stock solutions 0,05M

IAA (MW = 175 mol/g)
IAA powder stocked at -20°C
0,0876g in 10ml NaOH 0,1M
NAA (MW = 186 mol/g)
NAA powder stocked at room temperature
0,0931g in 10ml NaOH 0,1M

Stocked at 4°C

Day 1

Preparation of dilutions

Preparation of the mother solution
0,4ml IAA (or NAA) 0,05M
15ml MMR 0,1X
Ajust pH with HCl 1N
Fill up to the 20ml with MMR 0,1X

Solution n°1, 500uM :
7 ml mother solution + 7ml MMR 0,1X

Solution n°2, 250uM :
6ml solution n°1 + 6ml MMR 0,1X

Solution n°3, 125uM :
4ml solution n°2 + 4ml MMR 0,1X

Filling the wells with prepared solutions (~2ml/weel

12-wel plate 1 2 3 4
A MMR 0,1X Solution n°3 Solution n°2 Solution n°1
B MMR 0,1X Solution n°3 Solution n°2 Solution n°1
C MMR 0,1X Solution n°3 Solution n°2 Solution n°1

Add the embryos (see IVF protocol)

Add 10-30 embryos/well

Incubation

Incubator 21°C

Days after

Microscope observation

Take a picture of each well where it’s possible to see all the embryos
Count the alive and dead embryos
Remove the dead embryos

Medium changing

Protocol of DAY 1 for the preparation
Incline the plate and pipet the medium with a Pasteur pipette
Add MMR 0,1X in each well
Incline the plate and pipet the medium with a Pasteur pipette
Add the new medium following this map:


12-wel plate 1 2 3 4
A MMR 0,1X Solution n°3 Solution n°2 Solution n°1
A MMR 0,1X Solution n°3 Solution n°2 Solution n°1
A MMR 0,1X Solution n°3 Solution n°2 Solution n°1

Preparation of tadpole samples for HPLC

The original protocol from Vastag L, Jorgensen P, Peshkin L, Wei R, Rabinowitz JD, et al. (2011) Remodeling of the Metabolome during Early Frog Development. PLoS ONE 6(2): e16881. doi:10.1371/journal.pone.0016881 article was performed on 1 egg of Xenopus laevis. Because 1 embryo of this species represents 10 embryos of Xenopus tropicalis, we decided to perform our protocol on 10 Xenopus tropicalis embryos.

Aim: HPLC test to evaluate the presence of auxin in tadpoles embryos

Material

- IAA powder (stocked at -20°C)
- NAA powder (stocked at room temperature)
- Acetonitrile 100% HPLC grade
- Methanol 100% HPLC grade
- Falcon 15ml
- 1,5ml tubes
- Pasteur pipettes
- Mortar
- Centrifuge
- Ice
- Vortex
- P1000, P200 with tips

Prerequisite: To have perform a FIV and the auxin toxicity protocol the day before.
Before beginning: Turn on the centrifuge at 4°C

Preparation of solutions (under chemical hood)

Mix for 10 samples
2ml acetonitrile
2ml methanol
1ml H2O
Cool mix to -20oC
Prepare 1mg/ml auxin solutions in 20% acetonitrile/H2O


Preparation of the samples

Take 10 embryos from each concentration of IAA and NAA (0, 125, 250 and 500 µM)
Rinse the embryos 4 times in H2O
Place the embryos in 1,5ml tubes
Add 55µl of cold mix
Crush the embryos with a mortar
Vortex
Place on ice for 20min
Centrifuge 5min – 14000 rpm – 4°C (centrifuge eppendorf 5417R)
Collect as much as possible of the supernatant and place it in new tubes

To extract more:
Add 55µl of the mix on the pellet
Repeat all steps

Salkowski assay

Salkowski assay is the simplest way to test whether our samples (bacteria or tadpoles extracts)contain auxins.

Preparation of bacteria culture for assay

1. Measure the O.D. at 600nm of bacteria in order to observe difference in growth.
2. Centrifuge culture and filter supernatant through 0,2 µM filter.

Salkowski assay

1. Prepare the reagent:
Mix 0,5mL of FeCl3 0,5M with 25mL of HClO4 35%. Store in absence of light.
2. Add Salkowski reagent to the filtered supernatant in a ratio 2:1.
3. Incubate in dark for 30min.
4. Measure the D.O. at 530nm.











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