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Team:TU Darmstadt/Labjournal/Transport
From 2012.igem.org
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
- 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
- Restriction digest of PCR products by 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)
- 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
- 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)
