Difference between revisions of "Team:Toulouse/Experiments"

Revision as of 15:16, 1 September 2015

iGEM Toulouse 2015

YETM 500mL	TFB1 200mL	TFB2 200mL
2.5g Yeast Extract 10g Tryptone 5g MgSO4.7H2O Adjust pH to 7.5 with KOH For Plates: add 7.5g of Agar	0.59g KOAc 2.42g RbCl 0.29g CaCl2.2H2O 1.98g MnCl2.4H2O Adjust to pH 5.8 with 0.2 M acetic acid Add dH2O to 200mL Filter sterilize Store refrigerated at 4°C	0.42g MOPS 2.21g CaCl2.2H20 0.24g RbCl 30g Glycerol Adjust to pH 6.5 with KOH Add dH2O to 200mL Filter sterilize Store refrigerated at 4°C

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

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

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

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

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

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

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
Inactivation of the enzymes for the vector

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

References

@@ Line 303: / Line 303: @@
 <h3 style="font-size:20px;">Migration and gel extraction</h3>
 </div></center>
-<div class="group center">
+<div class="group">
+<ul align="justify" style="font-size:15px;">
+			<li>1. Prepare a 1% or 2% electrophoresis agarose gel with 0.5x TAE buffer </li>
+			<li>2. Put 20 µL of sample + 6 µL of marker (1 kb for 1% gel and 100 pb for 2%) into the well</li>
+			<li>3. Migration for 30 min at 100 V or 1 hour at 50 V</li>
+			<li> 4. Bathe 10 minutes in BET</li>
+			<li> 5. Wash in water for 5 minutes </li>
+			<li> 6. The gel extraction is realized thanks to the QIAGEN Gel Extraction Kit</li>
+			<li> </li>
+			<li> Inactivation of the enzymes for the vector</li>
+ </ul>
 </div>
 	<div class="subtitle" >