Team:Exeter/lab book/gibs/wk2

From 2012.igem.org

(Difference between revisions)
Line 91: Line 91:
       </font>
       </font>
-
     <!------------ENTER LAB BOOK HERE: PLEASE INCLUDE DATES WITHIN THE WEEK------------>  
+
     <!<p>16.7.12</p>
 +
<p>Protocol as on 11.7.12 (basic transformation procedure.)</p>
 +
<p>TetrRBS and RBS bio brick sequences resuspended in 10µl water</p>
 +
<p>Resuspended DNA (1µl) added to top10 competent cells (25µl)</p>
 +
<p>Incubated on ice for 30 minutes</p>
 +
<p>42 °C heat shock for 30 seconds</p>
 +
<p>Incubated on ice for 2 minutes</p>
 +
<p>S.O.C. Medium aseptically added (250µl)</p>
 +
<p>Incubated in shaking incubator - 37 C, 220 rpm, 1 hour</p>
 +
<p>LB(amp) (1:1000) plates made</p>
 +
<p>Spread plates of 20µl and 100µl transformed E. coli made.</p>
 +
<p>Overnight incubation at 37 °C</p>
 +
 
 +
<p>17.7.12</p>
 +
<p>Made up LB-agar for later use</p>
 +
<p>Chose 3 colonies from each 100 µl spread plate.</p>
 +
<p>Colonies transferred by pipette tip into 5 ml LB(amp) broth (1:1000 ratio)</p>
 +
<p>Incubated at 37 °C, 220 rpm, overnight</p>
 +
 
 +
<p>18.7.12 (miniprep attempt)</p>
 +
<p>Centrifuged tubes, 4℃, 10 minutes, 3901 rcf</p>
 +
<p>Pellet was considered too small, probably due to shaking incubator turning off</p>
 +
<p>4 colonies were selected from plates, put in broth and then incubated (as in 17.7.12)</p>
 +
 
 +
<p>19.7.12 (miniprepping)</p>
 +
<p>Transferred cultures to identical falcon tubes to remove pipette tips</p>
 +
<p>Centrifuged at 3901 rcf for 5 minutes, 4 °C</p>
 +
<p>Supernatant tipped away</p>
 +
<p>250µl resuspension solution added</p>
 +
<p>Pellet resuspended by gentle pipetting up and down</p>
 +
<p>250 µl lysis solution added</p>
 +
<p>Tubes inverted</p>
 +
<p>350 µl neutralisation solution rapidly added</p>
 +
<p>Tubes inverted</p>
 +
<p>Centrifuged at 13000rpm, 5 minutes, 4 °C</p>
 +
<p>Supernatant transferred to geneJET miniprep column</p>
 +
<p>Centrifuged at 13000rpm, 1 minute</p>
 +
<p>Flow through discarded</p>
 +
<p>500 µl wash solution added</p>
 +
<p>Centrifuged at 13000rpm, 30 seconds</p>
 +
<p>Discard flow through</p>
 +
<p>500 µl wash solution added</p>
 +
<p>Centrifuged at 13000rpm, 1 minute</p>
 +
<p>Discard flow through</p>
 +
<p>Centrifuged at 13000rpm, 1 minute</p>
 +
<p>Transferred column to a new 1.5 ml eppendorf</p>
 +
<p>50 µl elution buffer added</p>
 +
<p>Incubated for 2 minutes, room temperature</p>
 +
>  
      
      
     </font>
     </font>

Revision as of 22:28, 25 July 2012

ExiGEM2012 Lab Book Gibs wk2

Operon Construction: 16th - 20th July 2012

 16.7.12

Protocol as on 11.7.12 (basic transformation procedure.)

TetrRBS and RBS bio brick sequences resuspended in 10µl water

Resuspended DNA (1µl) added to top10 competent cells (25µl)

Incubated on ice for 30 minutes

42 °C heat shock for 30 seconds

Incubated on ice for 2 minutes

S.O.C. Medium aseptically added (250µl)

Incubated in shaking incubator - 37 C, 220 rpm, 1 hour

LB(amp) (1:1000) plates made

Spread plates of 20µl and 100µl transformed E. coli made.

Overnight incubation at 37 °C

17.7.12

Made up LB-agar for later use

Chose 3 colonies from each 100 µl spread plate.

Colonies transferred by pipette tip into 5 ml LB(amp) broth (1:1000 ratio)

Incubated at 37 °C, 220 rpm, overnight

18.7.12 (miniprep attempt)

Centrifuged tubes, 4℃, 10 minutes, 3901 rcf

Pellet was considered too small, probably due to shaking incubator turning off

4 colonies were selected from plates, put in broth and then incubated (as in 17.7.12)

19.7.12 (miniprepping)

Transferred cultures to identical falcon tubes to remove pipette tips

Centrifuged at 3901 rcf for 5 minutes, 4 °C

Supernatant tipped away

250µl resuspension solution added

Pellet resuspended by gentle pipetting up and down

250 µl lysis solution added

Tubes inverted

350 µl neutralisation solution rapidly added

Tubes inverted

Centrifuged at 13000rpm, 5 minutes, 4 °C

Supernatant transferred to geneJET miniprep column

Centrifuged at 13000rpm, 1 minute

Flow through discarded

500 µl wash solution added

Centrifuged at 13000rpm, 30 seconds

Discard flow through

500 µl wash solution added

Centrifuged at 13000rpm, 1 minute

Discard flow through

Centrifuged at 13000rpm, 1 minute

Transferred column to a new 1.5 ml eppendorf

50 µl elution buffer added

Incubated for 2 minutes, room temperature

>

Retrieved from "http://2012.igem.org/Team:Exeter/lab_book/gibs/wk2"