Team:UGent/Labjournal
From 2013.igem.org
(Difference between revisions)
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<li> <b>CcdB operon</b>: | <li> <b>CcdB operon</b>: | ||
<br> -> HiFi PCR of plasmids p5SpFRT-T7ccdB, p10SpFRT-T7ccdB and p20SpFRT-T7ccdB with MDM0586/MDM0587 to amplify ccdB operon | <br> -> HiFi PCR of plasmids p5SpFRT-T7ccdB, p10SpFRT-T7ccdB and p20SpFRT-T7ccdB with MDM0586/MDM0587 to amplify ccdB operon | ||
- | <br> -> Purification of CcdB operon PCR fragment using Qiagen Qiaquick PCR purification kit and checked on analytical gel: nanodrop | + | <br> -> Purification of CcdB operon PCR fragment using Qiagen Qiaquick PCR purification kit and checked on analytical gel (expected fragment of 2300 bp): nanodrop |
<br> p5: 174,6 ng/µl | <br> p5: 174,6 ng/µl | ||
<br> p10: 24,5 ng/µl (consistent with small band on the analytival gel) | <br> p10: 24,5 ng/µl (consistent with small band on the analytival gel) | ||
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<li> Inoculation colonies of pSB4A5 (plate 2, well 2J) and pSB6A1 (plate 2, well 2L) (2 times: one for further work and one for cryovials) </li> | <li> Inoculation colonies of pSB4A5 (plate 2, well 2J) and pSB6A1 (plate 2, well 2L) (2 times: one for further work and one for cryovials) </li> | ||
<li> Purification of plasmids pSB3T5, pSB6A1 and pSB4A5 </li> | <li> Purification of plasmids pSB3T5, pSB6A1 and pSB4A5 </li> | ||
- | <li> Generation of <b>restriction</b> digest fragments of T7-ccdB insert (from p5SpFRT-T7ccdB and p20SpFRT-T7ccdB) and vector (pSB3T5 (2x), pSB4A5 (from plate 2, well 2J) and pSB6A1 (1x from plate 5, well 1K and 1x from plate 2, well 2L) with XbaI and PstI-HF (gel purificy RD-fragments using Qiagen Qiaquick gel extraction kit): Nanodrop | + | <li> Generation of <b>restriction</b> digest fragments of T7-ccdB insert (from p5SpFRT-T7ccdB and p20SpFRT-T7ccdB) and vector (pSB3T5 (2x), pSB4A5 (from plate 2, well 2J) and pSB6A1 (1x from plate 5, well 1K and 1x from plate 2, well 2L) with XbaI and PstI-HF (gel purificy RD-fragments using Qiagen Qiaquick gel extraction kit (expected fragment of 2157 bp for T7-ccdB, 3229 bp for pSB3T5, 3372 bp for pSB4A5 and 3999 bp for pSB6A1): Nanodrop |
<br>p5: 43,7 ng/µl | <br>p5: 43,7 ng/µl | ||
<br>p20: 24,2 ng/µl | <br>p20: 24,2 ng/µl | ||
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<br> -> Plates with transformants: pSB6A1 some colonies, pSB4A5 some colonies and pSB3T5 no colonies </li> | <br> -> Plates with transformants: pSB6A1 some colonies, pSB4A5 some colonies and pSB3T5 no colonies </li> | ||
<li>Gel elektroforesis on RD-fragments from plasmids: show expected fragments</li> | <li>Gel elektroforesis on RD-fragments from plasmids: show expected fragments</li> | ||
- | <li><b>cPCR</b> on the pSB4A5 (primers: MDM0095 and CLG0019) and pSB6A1 (primers: MDM0096 and CLG0019) colonies with Taq polymerase: one <b>positive</b> colony of pSB6A1-T7ccdB (fragment of 437 bp) | + | <li><b>cPCR</b> on the pSB4A5 (primers: MDM0095 and CLG0019) and pSB6A1 (primers: MDM0096 and CLG0019) colonies with Taq polymerase: one <b>positive</b> colony of pSB6A1-T7ccdB (fragment of 437 bp)</li> |
+ | <li>Plate <b>transformation mixtures</b> again on glucose plates and grow overnight at 30°C | ||
+ | <br>->Plates with transformants: pSB4A5 colonies, pSB3T5 no colonies</li> | ||
</ul> | </ul> | ||
<br> | <br> |
Revision as of 18:08, 4 September 2013
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