Team:TU Darmstadt/Labjournal/Transport

From 2012.igem.org

(Difference between revisions)
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* Third approach to isolate the five genes from ''Comamonas t.'' by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Colony_PCR colony-PCR]
* Third approach to isolate the five genes from ''Comamonas t.'' by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Colony_PCR colony-PCR]
* PCR product purification using the Promega-Kit: ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K808005 tctB 197aa] (2), [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808002 tctA_503aa] (3), [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808003 tctB 162aa] (4), [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808001 tphC 322aa] (5))
* PCR product purification using the Promega-Kit: ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K808005 tctB 197aa] (2), [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808002 tctA_503aa] (3), [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808003 tctB 162aa] (4), [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808001 tphC 322aa] (5))
 +
:https://static.igem.org/mediawiki/2012/f/fc/W2_1_kolo_pcr_2_s19.png
 +
:(1): no PCR-product
 +
* Nanodrop measurements of the purified PCR products:
 +
:(2) : 56.1 ng/µl 260/280=1.87
 +
:(3) : 66.8 ng/µl 260/280=1.92
 +
:(4) : 72.8 ng/µl 260/280=1.87
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:(5) : 68.8 ng/µl 260/280=1.82
 +
 +
===week 3 (04.-08.06.12)===
 +
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Restriction_digest Restriction digest] of the purified PCR products and of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808004 tctA 505aa] (leftover of the 1. week) by SpeI and EcoRI
 +
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Restriction_digest Restriction restriction digest] of pSB1A2 using SpeI and EcoRI
 +
* Dephosphorylation of the restriction reactions by using antarctic Phosphatase
 +
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/DNA_Ligation Ligation] of the five genes into pSB1A2 and subsequently [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Bacterial_transformation transformation] into DH5α (incubation at 37°C)

Revision as of 21:33, 8 September 2012

Contents

Transport

Week 1 (21.-25.05.12)

  • First colony PCR of Comamonas testosteroni for isolation of the following genes: [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808004 tctA 505aa] (1), [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808005 tctB 197aa] (2), [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808002 tctA_503aa] (3), [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808003 tctB 162aa] (4), [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808001 tphC 322aa] (5). Analysis of [http://en.wikipedia.org/wiki/Polymerase_chain_reaction PCR] products by agarose gel electrohporesis
W1_1_kolo_pcr.png
  • PCR product purification using the Promega-Kit
  • Nanodrop measurements of the purified PCR products:
(1) : 19.5 ng/µl 260/280=1.58
(2) : 61.9 ng/µl 260/280=1.75
(3) : 75.1 ng/µl 260/280=1.75
(4) : 93.3 ng/µl 260/280=1.85
(5) : 91.3 ng/µl 260/280=1.58
W1_2_restriktionsverdau_s8.png
  • Purification of the bands gained form gel electrophoresis (Promega-Kit)
  • Nanodrop measurements of the purified products:
(1) : 8.3 ng/µl 260/280=1.94
(2) : 8.9 ng/µl 260/280=1.80
(3) : 13.0 ng/µl 260/280=1.57
(4) : 1.5 ng/µl 260/280=3.08
(5) : 3.7 ng/µl 260/280=2.26

week 2 (28.05.-01.06.12)

  • Purified products from 1. week were ligated into pSB1A2 and subsequently transformed into DH5α (in spite of low concentration)
  • Overnight incubation on LB agar plates; no growth detectable
  • Second approach to isolate the five genes from Comamonas t. using colony-PCR
  • Analysis of PCR products by agarose gel electrohporesis; without results; modification of the PCR protocols: adding DMSO
  • Third approach to isolate the five genes from Comamonas t. by colony-PCR
  • PCR product purification using the Promega-Kit: ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K808005 tctB 197aa] (2), [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808002 tctA_503aa] (3), [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808003 tctB 162aa] (4), [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808001 tphC 322aa] (5))
W2_1_kolo_pcr_2_s19.png
(1): no PCR-product
  • Nanodrop measurements of the purified PCR products:
(2) : 56.1 ng/µl 260/280=1.87
(3) : 66.8 ng/µl 260/280=1.92
(4) : 72.8 ng/µl 260/280=1.87
(5) : 68.8 ng/µl 260/280=1.82

week 3 (04.-08.06.12)

  • Restriction digest of the purified PCR products and of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808004 tctA 505aa] (leftover of the 1. week) by SpeI and EcoRI
  • Restriction restriction digest of pSB1A2 using SpeI and EcoRI
  • Dephosphorylation of the restriction reactions by using antarctic Phosphatase
  • Ligation of the five genes into pSB1A2 and subsequently transformation into DH5α (incubation at 37°C)
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