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Team:Harvard/color/notebook
From 2010.igem.org
abstract lab notebook pigment metabolism engineering pathways targeting the petals parts & primers results references
notebook
| Week 6 | - | - | - | - | 07-23-2010 |
| Week 7 | 07-26-2010 | 07-27-2010 | 07-28-2010 | 07-29-2010 | 07-30-2010 |
07-23-2010 [top]
- Received primers for LUT2 and BETA-OHASE 1 - began building knockdown constructs
- PCR reaction on RS300 to insert recognition sites. Primers listed below
- LUT2 rxn 1: A, CP4
- LUT2 rxn 2: CP2, CP3
- LUT2 rxn 3: CP1, B
- BETA-OHASE 1 rxn 1: A, CP8
- BETA-OHASE 1 rxn 2: CP6, CP7
- BETA-OHASE 1 rxn 3: CP5, B
- PCR purified completed reaction
07-26-2010 [top]
- Ran 4 ul of PCR reaction on a 1% agarose gel
- Bands appeared to be the correct size, so started PCR stitching process
- Ran two PCR reactions, one for each of LUT2 and BETA-OHASE 1
- Template: Rxn 1, 2, and 3 of the appropriate construct
- Template diluted to 1 ng/ul, used 1 ul of each template
- Primers: A and B for both reactions
- Size expected to be around 450 bp
- PCR purified and ran 4 ul on a 1% agarose gel
- PCR was not clean, decided to re-do stitching reaction with undiluted template (concentrations around 50 ng/ul, used 1 ul of each
- Ran 4 ul on 1% agarose gel
- Observed clear band at correct location for BETA-OHASE 1, but not for LUT2
[click to enlarge]
[click to enlarge]
[click to enlarge]07-27-2010 [top]
- Redid PCR stitching for LUT2, ran 4 ul on a gel
- Observed clear bands, including one in the right location
- Ran entire reaction on a gel (half in each lane), cut out appropriate band and gel purified
[click to enlarge]
[click to enlarge]
