Team:TU Darmstadt/Labjournal/Transport

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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)
  • colony-PCR of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808004 tctA 505aa] (1), 2 approaches
  • Analysis of PCR products by agarose gel electrohporesis. Only one of them showed the expected band. The band was excluded and purified via the Promega-Kit.
W3_1_kolo_pcr_505_s23.png
  • Nanodrop measurement of the purified product:
(1.1) =8.9 ng/µl 260/280=2,22
Because of low concentration and contamination the PCR was repeated and the product was purified again by the Promega-Kit.
  • Performing a colony-PCR-screen of the transformed cells using the Taq-Polymerase. Verification of 4 colonies per plate.
PCR did not work
  • Testing the Taq-Polymerase for function. The Phu-Polymerase was used for comparison. Only the approach using the Phu-Polymerase showed the expected bands in agarose gel electrohporesis.
W3_2_taq-phu_s27.2.png
  • Screening was repeated with Phu-Polymerase; no bands visible

week 4 (11.-15-06.12)

  • Inoculation of LB-media with DH5α_pSB1A2 and DH5α_pSB1C3; overnight incubation at 37°C
  • Miniprep of DH5α_pSB1A2 and DH5α_pSB1C3 using the Promega-Kit
  • Nanodrop measurements of the preparation:
pSB1A2 : 136.1 ng/µl 260/280= 1.93
pSB1C3 : 265.1 ng/µl 260/280= 1.89
W4_1_restriktion_der_vektoren.png
pSB1C3 shows several bands (Insert, vector without insert, linearized vector with insert); concentration of pSB1A2 is very low, only one band visible
  • Approach to isolate the following genes from Comamonas t. by colony-PCR: [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) and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808001 tphC 322aa] (5) by [http://en.wikipedia.org/wiki/Polymerase_chain_reaction PCR]
  • Analysis of PCR products by agarose gel electrohporesis
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