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LB medium

10 g Trypton
5 g yeast extract
10 g NaCl
12 g Agar-Agar (for plates)
Adjust pH to 7.4

TAE buffer

1 L of 50x TAE buffer
242.48 g Tris
41.02 g sodium acetate
18.612 g EDTA
Adjust pH to 7.8 with acetic acid
Solve in dH2O
Dilute 20 mL 50x stock in 1L dH2O for 1x Buffer for PAGE

M9 medium

To prepare 1 liter of M9 minimal medium add the following components to 836.7 mL sterile distilled water.
To avoid precipitation, start with CaCl2 and mix the solution thoroughly after addition of a component.
100 mL 10 X M9 salt solution
50 mL 20 X carbon source
10 mL 100 X trace element solution
1 mL autoclaved 1 M MgSO4
0.3 mL autoclaved 1 M CaCl2
1 mL filter sterilized 1 g/L biotin
1 mL filter sterilized 1 g/L thiamin

M9 salt stock solution

75.2 g Na2HPO4 x 2H2O
30 g KH2PO4
5 g NaCl
5 g NH4Cl
Dissolve the salts in 800 mL water and adjust the pH to 7.2 with NaOH. Add water to a final volume of 1 L and autoclave for 15 minutes at 121 °C.

Colony PCR

Pick one colony with a sterile tip and elute it in 100 µL ddH20 or medium
Store the colony in 4 °C while colony PCR is running
One reaction mix contains:

10 µL 5x buffer
2 µL MgCl2 (25 mM stock)
1 µL dNTPs
0.5 µL primer mix (prefix/suffix primers or sequencing primers)
35.25 µL ddH2O
0.25 µL GoTaq polymerase (Promega)
1 µL template

PCR program:

Start: 3 min, 98 °C
30 cycles of:

30 s, 98 °C
30 s, 55 °C
30 s / 1 kb template, 72 °C

Finish: 5 min, 72 °C

Gel electrophoresis: check the fragment size
Plate the correct colony

Generating electrocompetent cells

Material:

550 mL LB-Medium
1 L cooled bidest. H2O
150 mL cooled 10 % glycerine
10 pre-cooled 50 mL Falcons

Protocol:

Inoculate 2x3 mL LB with bacterial stock; incubate over night at 37 °C and 200 rpm
Inoculate 2x250 mL LB with the over night cultures in 1-litre-flask at 37 °C and 140 rpm
Incubate until OD600 0,4-0,6
Cool the culture 15-30 minutes on ice
Onwards all steps at 4°C
Divide the cultures into cooled 50 mL Falcons and centrifugate at 4000 rpm, 4 °C for 15 minutes,
make sure to slowly accelerate and deccelerate
Discard supernatant
Resuspend pellet in 5 mL cooled bidest H2O
(and don't get frustrated while doing it, keep shaking gently)
Pool two suspensions each, add bidest H2O up to 50 mL and centrifugate again
(see centrifugation above)
Discard supernatant
Resuspend pellet in 5 mL cooled bidest H2O
Add bidest H2O up to 50 mL and centrifugate again (see centrifugation above)
Discard supernatant
Resuspend pellet in 5 mL cooled 10 % glycerine
Transfer suspensions in two 50 mL Falcons and centrifugate again (see centrifugation above)
Discard supernatant
Add volume of 10 % glycerine that is approximately equal to the volume of the pellet and resuspend
Divide cells in 50 µL aliquots and freeze in liquid N2 immediately
Store at -80 °C

Transformation via electroporation

Thaw 50 µL competent E.coli cells on ice, dilute with icecold 50 µL glycerine (10 %) if necessary
Add 0.5-5 µL plasmid to 50 µl electrocompetent cells
Store cells on ice for 1 minute
Electroporate at U = 2.5 kV, C = 25 µF, R = 400 ?
Transfer transformation reaction to 450 µL SOC-Medium and shake 1 h at 37 °C
Centrifuge 2 min at 2000 rpm and plate on selective LB-Medium
Incubate over night at 37 °C

SOC-medium

Add the following components for 900 ml of distilled H2O:

20 g Trypton
5 g Bacto Yeast Extract
2 mL of 5 M NaCl
2.5 ml of 1 M KCl
10 ml of 1 M MgCl2
10 ml of 1 M MgSO4
20 ml of 1 M glucose

50X TAE Stock Solution

For each litre of solution:

242 g Tris Base (MW=121.1)
57.1 mL Glacial Acetic Acid
100 mL 0.5 M EDTA

mix Tris with stir bar to dissolve in about 600 mL of ddH2O.
add the EDTA and Acetic Acid.
bring final volume to 1 L with ddH20.
store at room temperature.

Note: Final (1x) working concentration:

0.04 M Tris - Acetate
0.001 M EDTA

50X Modified TAE Stock Solution

For each litre of solution:

242 g Tris Base (MW=121.1)
57.1 mL Glacial Acetic Acid
10 mL 0.5 M EDTA

mix Tris with stir bar to dissolve in about 600 mL of ddH2O.
add the EDTA and Acetic Acid, pH to 8.0.
bring final volume to 1 L with ddH2O.
store at room temperature.

Note: Final (1x) working concentration:

0.04 M Tris - Acetate
0.0001 M EDTA

DNA Purification by Centrifugation

Dissolving the Gel Slice

Following electrophoresis, excise DNA band from gel and place gel slice
in a 1.5 mL microcentrifuge tube.
Add 10 µL Membrane Binding Solution per 10 mg of gel slice. Vortex and
incubate at 50-65°C until gel slice is completely dissolved.

Processing PCR Amplifications

Add an equal volume of Membrane Binding Solution to the PCR amplification.

Binding of DNA

Insert SV Minicolumn into Collection Tube.
Transfer dissolved gel mixture or prepared PCR product to the Minicolumn
assembly. Incubate at room temperature for 1 minute.
Centrifuge at 16,000 x g for 1 minute. Discard flowthrough and reinsert Minicolumn
into Collection Tube.

Washing

Add 700 µL Membrane Wash Solution (ethanol added). Centrifuge at 16,000 x g
for 1 minute. Discard flowthrough and reinsert Minicolumn into Collection Tube.
Repeat Step before with 500 µL Membrane Wash Solution. Centrifuge at 16,000 x g for 5 minutes.
Empty the Collection Tube and recentrifuge the column assembly for 1 minute with the
microcentrifuge lid open (or off) to allow evaporation of any residual ethanol.

Elution

Carefully transfer Minicolumn to a clean 1.5 mL microcentrifuge tube.
Add 15 µL of Nuclease-Free Water to the Minicolumn. Incubate at 60°C
for 5 minutes. Centrifuge at 16,000 x g for 1 minute. Repeat this step.
Discard Minicolumn and store DNA at 4°C or -20°C.

Purification Protocol

Note:

All purification steps should be carried out at room temperature.
All centrifugation should be carried out in a table-top microcentrifuge at >12000 x g
(10 000-14 000 rpm, depending on the rotor type).

Resuspend the pelleted cells in 250 µL of the Resuspension Solution. Transfer the cell
suspension to a microcentrifuge tube. The bacteria should be resuspended completely by
vortexing or pipetting up and down until no cell clumps remain.

Note Ensure RNase A has been added to the Resuspension Solution.

Add 250 µL of the Lysis Solution and mix thoroughly by inverting the tube 4-6 times until
the solution becomes viscous and slightly clear.

Note Do not vortex to avoid shearing of chromosomal DNA. Do not incubate
for more than 5 minutes to avoid denaturation of supercoiled plasmid DNA.

Add 350 µL of the Neutralization Solution and mix immediately and thoroughly by inverting the tube 4-6 times.

Note It is important to mix thoroughly and gently after the addition of the
Neutralization Solution to avoid localized precipitation of bacterial cell debris.
The neutralized bacterial lysate should become cloudly.

Centrifuge for 5 minutes to pellet cell debris and chromosomal DNA.
Transfer the supernatant to the supplied GeneJET spin coloumn by decanting or pipetting. Avoid
disburbing or transferring the white precipitate.
Centrifuge for 1 minute. Discard the flow-through and place the coloumn back into the same
collection tube.

Note Do not add bleach to the flow-through.

Add 500 µL of the Wash Solution (diluted with ethanol prior to first use) to the GeneJET spin
column. Centrifuge for 30-60 seconds and discard the flow-through. Place the column back into the
same collection tube.
Repeat the wash procedure (step before) using 500 µL of the Wash Solution.
Discard the flow-through and centrifuge for an additional 1 minute to remove
residual Wash Solution. This step is essential to avoid residual ethanol in plasmid preps.
Transfer the GeneJET spin column into a fresh 1.5 mL microcentrifuge tube (not included). Add
50 µL of the Elution Buffer to the center of GeneJET spin column membrane to elute the plasmide
DNA. Take care not to contact the membrane with the pipette tip. Incubate for 2 minutes at room temperature
and centrifuge for 2 minutes.

Note An additional elution step (optional) with Elution Buffer or water will recover
residual DNA from the membrane and increase the overall yield by 10-20%.
For elution of plasmids or cosmids >20 kb, prewarm Elution Buffer to 70°C before applying to silica
membrane.

Discard the column and store the purified plasmid DNA at -20°C.

@@ Line 6: / Line 6: @@
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+            a:active{color:#13220;}
-        --></style>
+--></style>
 <h1> Protocols </h1>
-    <body>
+<div class="element" style="margin:10px 10px 10px 10px; padding:10px 10px 10px 10px">
+  <div id="text">
          <div class="tab" id="LBmedium">
              <div class="show">
@@ Line 28: / Line 29: @@
              </div>
              <div class="hide">
-                 <a  style="font-size:24px" href="#"><p style="margin-left:42%">LB medium </p></a>
+                 <a  style="font-size:24px" href="#"><p style="margin-left:30%">LB medium </p></a>
              </div>
              <div class="content">
-		 <ul style="margin-left:40%; margin-right:30%" type="disc">
+		 <ul style="margin-left:30%; margin-right:30%" type="disc">
 		 <li> 10 g Trypton </li>
 		 <li> 5 g yeast extract </li>
@@ Line 41: / Line 42: @@
-	<div class="tab" id="TAEbuffer">
+ 	<div class="tab" id="TAEbuffer">
              <div class="show">
                  <a href="#TAEbuffer">TAE buffer</a>
              </div>
              <div class="hide">
-                  <a  style="font-size:24px" href="#"><p style="margin-left:42%">TAE buffer </p></a>
+                  <a  style="font-size:24px" href="#"><p style="margin-left:30%">TAE buffer </p></a>
              </div>
              <div class="content">
-                 <ul style="margin-left:40%; margin-right:30%" type="disc">
+                 <ul style="margin-left:30%; margin-right:30%" type="disc">
 		<li> 1 L of 50x TAE buffer </li>
 		<li> 242.48 g Tris </li>
@@ Line 60: / Line 62: @@
          </div>
-<div class="tab" id="M9medium">
+        <div class="tab" id="M9medium">
              <div class="show">
                  <a href="#M9medium">M9 medium</a>
              </div>
              <div class="hide">
-                  <a  style="font-size:24px" href="#"><p style="margin-left:42%">M9 medium </p></a>
+                  <a  style="font-size:24px" href="#"><p style="margin-left:30%">M9 medium </p></a>
              </div>
              <div class="content">
-                 <ul style="margin-left:40%; margin-right:30%" type="disc">
+                 <ul style="margin-left:30%; margin-right:30%" type="disc">
-<li>To prepare 1 liter of M9 minimal medium add the following components to 836.7 mL sterile distilled water. <br>
+                <li>To prepare 1 liter of M9 minimal medium add the following components to 836.7 mL sterile distilled  water. <br>
-    To avoid precipitation, start with CaCl2 and mix the solution thoroughly after addition of a component. </li>
+                    To avoid precipitation, start with CaCl2 and mix the solution thoroughly after addition of a  component. </li>
-<li> 100 mL 10 X M9 salt solution </li>
+                <li> 100 mL 10 X M9 salt solution </li>
-<li> 50 mL 20 X carbon source </li>
+                <li> 50 mL 20 X carbon source </li>
-<li> 10 mL 100 X trace element solution </li>
+                <li> 10 mL 100 X trace element solution </li>
-<li> 1 mL autoclaved 1 M MgSO4 </li>
+                <li> 1 mL autoclaved 1 M MgSO4 </li>
-<li> 0.3 mL autoclaved 1 M CaCl2 </li>
+                <li> 0.3 mL autoclaved 1 M CaCl2 </li>
-<li> 1 mL filter sterilized 1 g/L biotin </li>
+                <li> 1 mL filter sterilized 1 g/L biotin </li>
-<li> 1 mL filter sterilized 1 g/L thiamin </li> </ul>
+                <li> 1 mL filter sterilized 1 g/L thiamin </li> </ul>
-</div>
+            </div>
-</div>
+        </div>
-<div class="tab" id="M9saltstocksolution">
+        <div class="tab" id="M9saltstocksolution">
-<div class="show">
+            <div class="show">
-<a href="#M9saltstocksolution">M9 salt stock solution </a>
+                <a href="#M9saltstocksolution">M9 salt stock solution </a>
-</div>
+            </div>
-<div class="hide">
+            <div class="hide">
- <a  style="font-size:24px" href="#"><p style="margin-left:42%">M9 salt stock solution </p></a>
+                <a  style="font-size:24px" href="#"><p style="margin-left:30%">M9 salt stock solution </p></a>
-</div>
+            </div>
-<div class="content">
+            <div class="content">
-<ul style="margin-left:40%; margin-right:30%" type="disc">
+                <ul style="margin-left:30%; margin-right:30%" type="disc">
-<li> 75.2 g Na2HPO4 x 2H2O </li>
+                <li> 75.2 g Na2HPO4 x 2H2O </li>
-<li> 30 g KH2PO4 </li>
+                <li> 30 g KH2PO4 </li>
-<li> 5 g NaCl </li>
+                <li> 5 g NaCl </li>
-<li> 5 g NH4Cl </li>
+                <li> 5 g NH4Cl </li>
-<li> Dissolve the salts in 800 mL water and adjust the pH to 7.2 with NaOH. Add water to a final volume of 1 L and autoclave for 15 minutes at 121 °C. </li></ul>
+                <li> Dissolve the salts in 800 mL water and adjust the pH to 7.2 with NaOH. Add water to a final volume of 1 L and autoclave for 15 minutes at 121 °C. </li></ul>
-</div>
+           </div>
-</div>
+       </div>
 <div class="tab" id="ColonyPCR">
@@ Line 103: / Line 105: @@
 </div>
 <div class="hide">
-  <a  style="font-size:24px" href="#"><p style="margin-left:42%">Colony PCR </p></a>
+  <a  style="font-size:24px" href="#"><p style="margin-left:30%">Colony PCR </p></a>
 </div>
 <div class="content">
-<ul style="margin-left:40%; margin-right:30%" type="disc">
+<ul style="margin-left:30%; margin-right:30%" type="disc">
 <li> Pick one colony with a sterile tip and elute it in 100 µL ddH20 or medium </li>
 <li> Store the colony in 4 °C while colony PCR is running</li>
@@ Line 134: / Line 136: @@
 </div>
 <div class="hide">
-  <a  style="font-size:24px" href="#"><p style="margin-left:42%">Generating electrocompetent </p></a>
+  <a  style="font-size:24px" href="#"><p style="margin-left:30%">Generating electrocompetent </p></a>
 </div>
 <div class="content">
-<ul style="margin-left:40%; margin-right:30%" type="disc">
+<ul style="margin-left:30%; margin-right:30%" type="disc">
 <li><u> Material: </li></u>
 <ul> <li> 550 mL LB-Medium </li>
@@ Line 175: / Line 177: @@
 </div>
 <div class="hide">
-  <a  style="font-size:24px" href="#"><p style="margin-left:42%">Transformation via electroporation </p></a>
+  <a  style="font-size:24px" href="#"><p style="margin-left:30%">Transformation via electroporation </p></a>
 </div>
 <div class="content">
-<ul style="margin-left:40%; margin-right:30%" type="disc">
+<ul style="margin-left:30%; margin-right:30%" type="disc">
 <li> Thaw 50 µL competent E.coli cells on ice, dilute with icecold 50 µL glycerine (10 %) if necessary </li>
 <li> Add 0.5-5 µL plasmid to 50 µl electrocompetent cells </li>
@@ Line 194: / Line 196: @@
 </div>
 <div class="hide">
-  <a  style="font-size:24px" href="#"><p style="margin-left:42%">SOC-medium </p></a>
+  <a  style="font-size:24px" href="#"><p style="margin-left:30%">SOC-medium </p></a>
 </div>
 <div class="content">
-<ul style="margin-left:40%; margin-right:30%" type="disc">
+<ul style="margin-left:30%; margin-right:30%" type="disc">
 <li> Add the following components for 900 ml of distilled H2O: </li>
 <ul> <li>20 g Trypton </li>
@@ Line 214: / Line 216: @@
 </div>
 <div class="hide">
-<a  style="font-size:24px" href="#"><p style="margin-left:42%"> 50X TAE Stock Solution </p></a>
+<a  style="font-size:24px" href="#"><p style="margin-left:30%"> 50X TAE Stock Solution </p></a>
 </div>
 <div class="content">
-<ul style="margin-left:40%; margin-right:30%" type="disc">
+<ul style="margin-left:30%; margin-right:30%" type="disc">
 <li> For each litre of solution: </li>
 <ul> <li> 242 g Tris Base (MW=121.1) </li>
@@ Line 238: / Line 240: @@
 </div>
 <div class="hide">
-<a  style="font-size:24px" href="#"><p style="margin-left:42%"> 50X Modified TAE Stock Solution </p></a>
+<a  style="font-size:24px" href="#"><p style="margin-left:30%"> 50X Modified TAE Stock Solution </p></a>
 </div>
 <div class="content">
-<ul style="margin-left:40%; margin-right:30%" type="disc">
+<ul style="margin-left:30%; margin-right:30%" type="disc">
 <li> For each litre of solution: </li>
 <ul> <li> 242 g Tris Base (MW=121.1) </li>
@@ Line 263: / Line 265: @@
 </div>
 <div class="hide">
-<a  style="font-size:24px" href="#"><p style="margin-left:42%"> DNA Purification by Centrifugation </p></a>
+<a  style="font-size:24px" href="#"><p style="margin-left:30%"> DNA Purification by Centrifugation </p></a>
 </div>
 <div class="content">
-<ul style="margin-left:40%; margin-right:30%" type="disc">
+<ul style="margin-left:30%; margin-right:30%" type="disc">
 <li> Dissolving the Gel Slice </li>
 <ul> <li> Following electrophoresis, excise DNA band from gel and place gel slice <br>
@@ Line 302: / Line 304: @@
 </div>
 <div class="hide">
-<a  style="font-size:24px" href="#"><p style="margin-left:42%">Puricfication Protocol </p></a>
+<a  style="font-size:24px" href="#"><p style="margin-left:30%">Puricfication Protocol </p></a>
 </div>
 <div class="content">
-<ul style="margin-left:40%; margin-right:30%" type="disc">
+<ul style="margin-left:30%; margin-right:30%" type="disc">
 <li> Note: </li>
 <ul> <li> All purification steps should be carried out at room temperature. </li>
@@ Line 346: / Line 348: @@
 </div>
 </div>
-    </body>
+</div>
+</div>
 </html>

Team:Bielefeld-CeBiTec/Notebook/Protocols

From 2014.igem.org

Revision as of 16:21, 11 August 2014

Protocols