Team:UEA-JIC Norwich/Methods

(Difference between revisions)

Revision as of 11:19, 1 July 2011

1. Thaw a tube of NEB 5-alpha Competent E.coli cells on ice for 10 minutes

2. Mark the location of the Biobrick well (letters from top to bottom, numbers from left to right)

3. Resuspend specific biobrick part (1 μl) with distilled water (20 μl). Aspirate up and down a few times

4. Inoculate with DNA ( 1 μl) to the competent cells

5. Place the mixture on ice for 30 minutes. Do not mix

6. Heat shock at exactly 42°C for exactly 30 seconds. Do not mix

7. Place on ice for 5 minutes. Do not mix

8. Pipette 950 μl of room temperature SOC into the mixture

9. Place at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate

10. Warm selection plates to 37°C

11. Do serial dilutions (2-3) at 105. Mix cells, flick/invert gently

12. Spread (100 μl) on agar plates of each dilution

13. Incubate overnight at 37°C

1. Select a colony by sampling it with a wooden pick

2. Place into the appropriate antibiotic LB broth

3. Incubate at 37°C overnight in an Incubator Shaker

4. Store at -20°C

1. Take 500µL of a previously prepared culture and place into a screw-top eppendorf tube

2. Add 500µL of Glycerol solution (V.V 50% to make a 25% concentration

3. Place in -20°C storage

@@ Line 132: / Line 132: @@
 <p>13. Incubate overnight at 37°C</p>
+<h2>Culture Preparation Protocol</h2>
+<p>1. Select a colony by sampling it with a wooden pick</p>
+<p>2. Place into the appropriate antibiotic LB broth</p>
+<p>3. Incubate at 37°C overnight in an Incubator Shaker</p>
+<p>4. Store at -20°C</p>
+<h2>Glycerol Stock Solution Protocol</h2>
+<p>1. Take 500µL of a previously prepared culture and place into a screw-top eppendorf tube</p>
+<p>2. Add 500µL of Glycerol solution (V.V 50% to make a 25% concentration</p>
+<p>3. Place in -20°C storage</p>
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