Revision as of 10:50, 20 September 2012

UNAM-Genomics_Mexico

Protocols

E. Coli Protocols

E. coli Protocols

PCR Protocol (50µl)

Buffer Pfu 5 µl
Upper primers 2.5 µl
Lower primers 2.5 µl
MgCL2 1 µl
DNA (depends on concentration, usually 0.5µl)
Enzime Taq Polymerase 1 µl
DNTP’s 8 µl
Water 29.5 µl

Cycles :)
95ºC 4 min
95ºC 1 min
55ºC :30 secs
70ºC 1 min
goto 2:4 30 times
72ºC 5 min

Hot start PCR protocol

Preheated 105ºC
Heated Lid ON
Pause OFF
In denat 94ºC 5 min.
Hot start OFF

30 cycles
94 ºC 30 secs.
55ºC 30 secs.
70ºC 45 secs.
72ºC 5 min
hold 10ºC

Ligation

These are done in 1 mL eppendorf tubes and normally have a T.V. of 20 µl.

•Add A µl of the insert (plasmid digested with corresponding enzymes).
•Add B µl of vector (plasmid digested with corresponding enzymes and complementary so that the ligation can be done properly) and properly DEPHOSPOHATED.
•Add 2 µl T4 DNA ligase buffer.
•Add 20-A-B-3-1 µl H2O miliQ.
•Add 1 µl T4 DNA ligase enzyme.

Dephosphorylation protocol

-It is done to plasmids digested with the corresponding enzymes that will serve as a “vector” in a ligation.
1. Add 1 µl alkaline phosphatase.
2. Add 2 µl buffer.
3.. Add 20 µl of Digestion.
4. Leave 1 hour at 37ºC.
5. Inactivate at 70ºC for 10 minutes.

Transformation

•Pipette the plasmid to a 1.3 mL eppendorf with competent cells and mix with the pipette.
•Incubate tubes on ice for 42 minutes.
•Heatshock at 42ºC for 2 minutes.
•Incubate for 5 minutes on ice.
•Add 1 mL LB broth to each tube.
•Mix by inversion 2-3 times.
•Incubate at 37ºC for an hour with shaking.
•During this hour prepare LB agar plates (30 mL) containing the appropriate antibiotics.
•After the hour Plate 100 µl.
•Centrifuge at 10000 rpm for 2 minutes.
•Drain 700-800 µl of supernatant, mix pellet with the rest and plate. Add to the plate.
•Let grow overnight at 37ºC.

Liquid culture

•Once the bacteria has grown, add 4 mL LB broth to a glass tube.
•Add the appropriate antibiotic.
•With a wooden toothpick take one colony per tube. Place the toothpick in the tube and vortex.
•Incubate the tube in the shaker 37ºC for at least 6 hours.

Digestion protocol (20 µl)

H2O 9.5 µl
Enzyme 0.5 µl
Buffer 10x 2 µl
DNA (it depends on the concentration but we usually put 8 µl)
37ºC at least 4 hours
For double digestions add 0.5 µl of the other enzyme and 0.5 µl less of H2O.

Gel extraction protocol

(Fermentas Gene Jet Gel extraction kit)
The extraction and purification of a DNA band from a gel electrophoresis is done when we digest and want to recover a specific fragment.
1. Cut the band of the fragment you wish to purify.
2. 400 µl of Binding Buffer, 10 min. at 65ºC with shaker so that the agarose melts.
3. 200 µl deisopropanol after 5 min in the shaker.
4. Pass through column and centrifuge. Throw out supernatant.
5. 700 µl wash buffer, centrifuge, throw it out, centrifuge.
6. Pass column to clean tube, add 40 µl de elution buffer.

Lysis protocol

1. Cultivate strain of interest in 5 µl LB with antibiotics.
2. Centrifuge culture at 14000rpm 2 minutes. Remove supernatant and repeat.
3. Add 200 µl solution I, incubate 5 minutes at room temperature. Mix well to dissolve pellet.
4. Add 400 µl of solution II and incubate 5 minutes at room temperature. Mix by inversion 3-6 times.
5. Add 300 µl of solution III. .
6. Mix by inversion and leave in ice 10 minutes. .
7. Centrifuge 10 minutes at 14000 rpm. .
8. Place supernatant in clean tubes and add 500µl 100% ethanol. Centrifuge 15 minutes at 14000 rpm and throw away supernatant. .
9. Add 1 ml 70% ethanol, centrifuge 5 minutes at 1200 rpm and throw out supernatant. Do this twice. .
10. Dry in SAVANT 5 minutes. .
11. Resuspend in 50 µl water MQ and add 5 µl RNAse. .

Gel electrophoresis

-Sample preparation
• Add 10 µl of DNA in each well.
• Add 3 µl loading buffer in the same well
• There are special markers that contain a mix of molecules of known size to compare and determine the size of our sample.
• Add 1 µl marker (ladder).

-Gel preparation and loading
• Add 1% agarose to an electrophoresis tank and let it become a gel.
• Introduce the tank in the chamber.
• In each well, with the pipette mix the content and load it in its corresponding well-
• Run with the appropriate Voltage, miliampers (mA) and time, corresponding to the size.

-Visualization
• Once completed, remove gel from chamber and place in special plastic container.
• Dye with ethidium bromide (5 minutes approximately).
• Rinse 10 minutes approximately with water.
• Visualize with UV light.

Competent cells protocol

• Grow cells in LB all night.
• Dilute 100x in LB 50 µl and let grow for 2 hours till an OD of 0.45-0.55 (40 ml X 4 tubes).
• Leave 10 minutes on ice, centrifuge 10 minutes at 6000 rpm at 4ºC.
• Resuspend in 16ml of ice cold TfbI.
• Leave 5 minutes on ice, centrifuge as before.
• Resuspend in 1.6 ml of cold TfbII.
• Leave 10 minutes on ice.
• Make 200 microliters aliquots and store at -70ºC.

@@ Line 60: / Line 60: @@
 •Add 1 µl T4 DNA ligase enzyme. <br /><br />
 </p></td>
-</tr></table>
+</tr>
-<h2>Dephosphorylation protocol</h2><br />
+<tr>
+<td id="leftcolumn2"><p>
+<center><h2>'''Dephosphorylation protocol'''</h2></center><br />
 -It is done to plasmids digested with the corresponding enzymes that will serve as a “vector” in a ligation. <br />
 . Add 1 µl  alkaline phosphatase. <br />
@@ Line 69: / Line 71: @@
 . Leave 1 hour at 37ºC. <br />
 . Inactivate at 70ºC for 10 minutes. <br />
+</p></td>
+<td  id="contentcolumn2"><p>
+<center><h2>Transformation</h2></center><br />
+•Pipette the plasmid to a 1.3 mL eppendorf with competent cells and mix with the pipette. <br />
+•Incubate tubes on ice for 42 minutes. <br />
+•Heatshock at 42ºC for 2 minutes. <br />
+•Incubate for 5 minutes on ice. <br />
+•Add 1 mL LB broth to each tube. <br />
+•Mix by inversion 2-3 times. <br />
+•Incubate at 37ºC for an hour with shaking. <br />
+•During this hour prepare LB agar plates (30 mL) containing the appropriate antibiotics. <br />
+•After the hour Plate 100 µl. <br />
+•Centrifuge at 10000 rpm for 2 minutes. <br />
+•Drain 700-800 µl of supernatant, mix pellet with the rest and plate. Add to the plate. <br />
+•Let grow overnight at 37ºC. <br /><br />
+</p></td>
+<td id="rightcolumn2"><p>
-<h2>Transformation</h2><br />
+<center><h2>Liquid culture</h2></center><br />
-•	Pipette the plasmid to a 1.3 mL eppendorf with competent cells and mix with the pipette. <br />
+•Once the bacteria has grown, add 4 mL LB broth to a glass tube. <br />
-•	Incubate tubes on ice for 42 minutes. <br />
+•Add the appropriate antibiotic. <br />
-•	Heatshock at 42ºC for 2 minutes. <br />
+•With a wooden toothpick take one colony per tube. Place the toothpick in the tube and vortex. <br />
-•	Incubate for 5 minutes on ice. <br />
+•Incubate the tube in the shaker 37ºC for at least 6 hours. <br /><br />
-•	Add 1 mL LB broth to each tube. <br />
+</p></td>
-•	Mix by inversion 2-3 times. <br />
+</tr>
-•	Incubate at 37ºC for an hour with shaking. <br />
+</table>
-•	During this hour prepare LB agar plates (30 mL) containing the appropriate antibiotics. <br />
-•	After the hour Plate 100 µl. <br />
-•	Centrifuge at 10000 rpm for 2 minutes. <br />
-•	Drain 700-800 µl of supernatant, mix pellet with the rest and plate. Add to the plate. <br />
-•	Let grow overnight at 37ºC. <br /><br />
-<h2>Liquid culture</h2><br />
-•	Once the bacteria has grown, add 4 mL LB broth to a glass tube. <br />
-•	Add the appropriate antibiotic. <br />
-•	With a wooden toothpick take one colony per tube. Place the toothpick in the tube and vortex. <br />
-•	Incubate the tube in the shaker 37ºC for at least 6 hours. <br /><br />
 <h2>Digestion protocol (20 µl)</h2><br />
 H2O 9.5 µl<br />

Team:UNAM Genomics Mexico/Notebook/Protocols

From 2012.igem.org