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Team:Harvard/color/notebook
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<span id="date"><a name="08-06-2010" class="date">08-06-2010</a></span> [<a class="top" href="#notebook">top</a>]<hr/> | <span id="date"><a name="08-06-2010" class="date">08-06-2010</a></span> [<a class="top" href="#notebook">top</a>]<hr/> | ||
| + | <ul> | ||
| + | <li>Got colonies for C3 (LYC) but not for C2 (BETA-OHASE 1). Picked four colonies and set up cultures for C3</li> | ||
| + | <li>Redid digestion on C2, and digested PCR products C4-6 with EcoRI and PstI. Also digested B21 with EcoRI and PstI with TSAP to obtain backbone.</li> | ||
<div><a href="https://static.igem.org/mediawiki/2010/4/45/080610pcrdigest1.png" id="single_image"><img src="https://static.igem.org/mediawiki/2010/4/45/080610pcrdigest1.png" width="100px" /> [click to enlarge]</a></div> | <div><a href="https://static.igem.org/mediawiki/2010/4/45/080610pcrdigest1.png" id="single_image"><img src="https://static.igem.org/mediawiki/2010/4/45/080610pcrdigest1.png" width="100px" /> [click to enlarge]</a></div> | ||
| - | < | + | <li>C2 and B21 digests worked, C4 had a PstI site inside the part and C5/C6 had EcoRI sites</li> |
| + | <li>Gel purified C2 and B21 (top) bands</li> | ||
| + | <li>Redigested C4-C6 with XbaI and SpeI, as well as B21 with XbaI and SpeI with TSAP to obtain backbone<li> | ||
<div><a href="https://static.igem.org/mediawiki/2010/a/a0/080610pcrdigest2.png" id="single_image"><img src="https://static.igem.org/mediawiki/2010/a/a0/080610pcrdigest2.png" width="100px" /> [click to enlarge]</a></div> | <div><a href="https://static.igem.org/mediawiki/2010/a/a0/080610pcrdigest2.png" id="single_image"><img src="https://static.igem.org/mediawiki/2010/a/a0/080610pcrdigest2.png" width="100px" /> [click to enlarge]</a></div> | ||
| - | < | + | <li>Gel ran strangely - ladder didn't show. Cut out bands and gel purified, then ran second gel to check sizes again</li> |
| - | + | ||
| + | <li>Miniprepped cultures set up in the morning, and sent out for sequencing</li> | ||
| + | </ul> | ||
</div> | </div> | ||
</html> | </html> | ||
Latest revision as of 03:43, 23 October 2010
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 |
| Week 8 | - | - | 08-04-2010 | 08-05-2010 | 08-06-2010 |
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
- Digested gel purified LUT2, PCR purified BETA-OHASE 1, and B21 (Biobrick part in V0120 - to serve as backbone) with EcoRI and PstI to prepare for ligation of constructs into biobrick backbone
- LUT2 had the correctly sized band, but BETA-OHASE 1 had two bands, one at 400 and one at 100.
- Checked sequences of primers, and determined that primers for BETA-OHASE 1 contained an EcoRI site
- Ordered new primers without biobrick restriction sites for BETA-OHASE 1. Checked other primers for restriction sites, and found them to be clean.
[click to enlarge]
[click to enlarge]
[click to enlarge]
07-28-2010 [top]
- Gel purified LUT2 and top band from B21 (backbone)
- Ligated LUT2 and backbone. Transformed into TURBO e. coli and plated on LB + Amp.
08-04-2010 [top]
- Received new primers for BETA-OHASE 1 - began building knockdown constructs for that and LYC
- PCR reaction on RS300 to insert recognition sites. Primers listed below
- LYC rxn 1: A, CP12
- LYC rxn 2: CP10, CP11
- LYC rxn 3: CP9, B
- BETA-OHASE 1 rxn 1: A, CP16
- BETA-OHASE 1 rxn 2: CP14, CP15
- BETA-OHASE 1 rxn 3: CP13, B
08-05-2010 [top]
PCR Stitching of LYC and BETA-OHASE 1 amiRNA parts; Digestion and Ligation into biobrick backbone
PCR Stitching of LYC and BETA-OHASE 1 amiRNA parts; Digestion and Ligation into biobrick backbone
- Ran PCR reaction on a 1% agarose gel.
- Initial PCR was successful. Gel purified parts
- Performed PCR Stitching for each of BETA-OHASE 1 and LYC
- Template: Rxn 1, 2, and 3 of the appropriate construct
- Used 1 ul of each template - concentrations were all around 25 ng/ul
- 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 successful. Gel purified products
- Digested products with EcoRI and PstI to ligate into V0120 backbone.
- Successful digestion - gel purified and ligated into backbone digested EcoRI/PstI last week
- Transformed into TURBO E. coli and plated on LB + Amp
[click to enlarge]
[click to enlarge]
[click to enlarge]- Ran PCR to amplify petal promoters
- Template: Arabidopsis genomic DNA, 80ng
- Primers: CP17/CP18 (At1G11850), CP19/CP20 (At1G70720 1.1kb), CP21/CP22 (At1G70720 1.5kb)
- Protocol: Phusion
- Ran completed reaction on 1% agarose gel
- PCR Failed. Retry with PFx
- Template: Arabidopsis genomic DNA, 80ng
- Primers: CP17/CP18 (At1G11850), CP19/CP20 (At1G70720 1.1kb), CP21/CP22 (At1G70720 1.5kb)
- Protocol: PFx, extension time 2 min
- Ran completed reaction on 1% agarose gel
[click to enlarge]
[click to enlarge]
08-06-2010 [top]
- Got colonies for C3 (LYC) but not for C2 (BETA-OHASE 1). Picked four colonies and set up cultures for C3
- Redid digestion on C2, and digested PCR products C4-6 with EcoRI and PstI. Also digested B21 with EcoRI and PstI with TSAP to obtain backbone.
- C2 and B21 digests worked, C4 had a PstI site inside the part and C5/C6 had EcoRI sites
- Gel purified C2 and B21 (top) bands
- Redigested C4-C6 with XbaI and SpeI, as well as B21 with XbaI and SpeI with TSAP to obtain backbone
- Gel ran strangely - ladder didn't show. Cut out bands and gel purified, then ran second gel to check sizes again
- Miniprepped cultures set up in the morning, and sent out for sequencing
[click to enlarge]
[click to enlarge]
