Team:Exeter/lab book/gibs/wk4
From 2012.igem.org
Jamesml213 (Talk | contribs) |
|||
Line 188: | Line 188: | ||
</table> | </table> | ||
+ | |||
+ | <table width="980" align="center" cellspacing="20"> | ||
+ | <tr align="center"> | ||
+ | <td> | ||
+ | <font color="#57B947" size="1" face="Verdana"> | ||
+ | <p><u>Website Designed and Built by: Ryan Edginton, James Lynch & Alex Clowsley</u> | | ||
+ | <a href="https://igem.org/Team.cgi?id=764" style="color:#57B947" target="_blank"><u>Contact Us</u></a> | | ||
+ | <a href="https://2012.igem.org/Team:Exeter/site_map" style="color:#57B947"><u>Site Map</u></a></p> | ||
+ | </font> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </table> | ||
</body> | </body> | ||
</html> | </html> |
Latest revision as of 00:09, 27 September 2012
Operon Construction: 30th July - 3rd August 2012 **Monday 30.7.12**4 pm Inoculated LB(kan) broth with transformed pBAD large (BBa_I0500) colonies according to protocol **Tuesday 31.7.12** 9 am Centrifuged broth tubes for 5 minutes, 3901 rcf, 4 °C Supernatant discarded Miniprep of pBAD (BBa_I0500) Step 4 changed to 9 minutes
Step 1020µl milli-Q water added in place of elution buffer, followed by another 10 µl after incubation and centrifugation 3A BioBrick Assembly for pBAD-RBS (BBa_I0500 + BBa_B0034) Digestion: Upstream pBAD (BBa_I0500) plasmid DNA: 8.68 µl MilliQ water: 18 µl Downstream RBS (BBa_B0034) plasmid DNA: 2.94 µl MilliQ water: 14.56 Destination plasmid (pSB1K3 – kanamycin resistance) digested according to protocol for linearised backbone Digested at 37 °C for 1 hour Heat inactivated at 80 °C for 20 minutes Ligation according to same protocol Downstream digest: 2 µl Plasmid digest: 2 µl Water: 2.5 µl Incubated at room temperature for 1.5 hours Heat inactivated at 80 °C for 20 minutes 2pm Sent DNA off for sequencing with VF2 and VR primers – pBAD (BBa_I0500) according to Bioline instructions 3 pm pBAD-RBS construct transformed 2 µl of construct DNA added to competent cells Plated volumes were 50 µl and 150 µl on LB(kan) **Thursday 2.8.12** 9 am Broth tubes centrifuged for 5 minutes, 3901 rcf, 4 °C Supernatant discarded Miniprep of pBAD-RBS (BBa_I0500 + BBa_B0034) Step 1045µl milli-Q water added in place of elution buffer, followed by another 5 µl after incubation and centrifugation 2.30 pm Gel of pPAD large-RBS (BBa_I0500 + BBa_B0034) Digest for gel (using only one enzyme – nanodrop data was uncertain) DNA (assumed ~100ng/µl): 5 µl EcoRI-HF: 1µl 100X BSA: 0.5 µl 10X NEBuffer 2: 5 µl Water: 11 µl Incubated at 37 °C for 20 minutes Run on 1% agarose gel LB(amp) streak plates made for pBAD-RBS (BBa_I0500 + BBa_B0034) dregs from miniprep. |
Website Designed and Built by: Ryan Edginton, James Lynch & Alex Clowsley | Contact Us | Site Map |