Team:TU Darmstadt/Labjournal/Transport

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), 50µl reaction mixture each; analysis of [http://en.wikipedia.org/wiki/Polymerase_chain_reaction PCR] products by agarose gel electrohporesis

• 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

• Digestion of PCR products by the restriction enzymes SpeI and EcoRI, heat inactivation after digestion
• Analysis of restriction digest by Agarose gel electrohporesis

• 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)