Team:TU Darmstadt/Labjournal/Transport

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Transport

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

(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

Restriction digest of PCR products by SpeI and EcoRI, heat inactivation after digestion
Analysis of restriction digest by agarose gel electrohporesis

(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

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))

(1): no PCR-product

(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

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.

(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.