Team:Cambridge/LabBook/Week4

From 2010.igem.org

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(→21. Experiment: Set up overnight cultures (E. coli/pHK555 and TOP10/pHK724) (Hannah & Will))
(→25. Results: Transformation of TOP10, BW25113 Δhns::kan and GM230 hns-205::Tn10 TetR with pHK555 and pHK724)
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No bacteria from any of the three strains took up both plasmids simultaneously, although when both the plasmids were added to a strain simultaneously there was growth on both of the individual resistance plates – i.e. some bacteria had taken up one and some the other but the probability that any would take up both was so low that no colonies were observed on the ChlAmp plate.
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No bacteria strains were killed by the heat shocking as smears of bacteria were seen on every plate without antibiotics added that also had a transformed strain plated.
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The takeup of plasmid 7 was successful in Invitrogen Top 10 strain and in the Red Strain.
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In the Black strain, the results from the Black strain are less clear – when we added both plasmids there was no colony growth on the Ampicillin plate (plasmid 7 indicates ampicillin resistance) therefore no plasmid 7 was taken up, however we observed growth on the plate which had been transformed by only plasmid 7 then added to an Amp plate. The colony size was extremely variable though, and although there was no colour differences visible we hypothesise that it may have been contaminated during the procedure.
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===26. Experiment: Streaking out of <i>E. coli</i>/pHK555 trasnformed with pHK724 (Paul, Emily & Anja)===
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Streaked out on a LB agar plate with Cm and Amp
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Used to write out 2x 'Cambridge', 'Sterilin' & 'iGEM 2010' on LB agar plates with Amp.
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===27. Experiment: of plasmids pHK724 & pHK555 from TOP10 & SLOCK 10 cells respectively (Will & Emily)===
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Followed "QIAprep Spin Miniprep Kit" protocl from QIAGEN
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4 ml of overnight cultures from each (prepared by Will & Hanah) of TOP10/pHK724 and <i>E. coli</i>/pHK555 were used to extract plasmids.
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====Results====
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50μl of sultion was obtained, 1μl of which was used to NanoDrop
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{| class="wikitable"
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|-
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! plasmid
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! Nucleic acid conc<sup>n</sup>
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|-
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| pHK555
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| 34.1ng/μl
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|-
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| pHK724
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| 15.7ng/μl
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|}
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plasmids were stored at 4°C
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===28. Experiment: Making long-term stocks of pHK555 (in Slock10) & pHK724 (in TOP10) (Peter & Emily)===
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====Preparation of 40% glycerol solution====
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{| class="wikitable"
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|-
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| 100% glycerol
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| 40ml
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|-
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| Deionized sterilised water
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| 60ml
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|}
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For pHK555 & pHK724, separately:
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* Added 1ml of 40% glycerol solution to cryogenic vial
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* Added 1ml of sample culture to vial
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* Gently vortexed vial to mix
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Stored in -80°C freezer in 'Black-GM230 hns-205' storage box. Vials are labelled with stickers: '7' for pHK724, '5' for pHK555.
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===29. Experiment: Transformation of TOP10, Red strain and Black strain with plasmid pHK724. They have each already been transformed with plasmid pHK555 (Hannah & Theo)===
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====Aim====
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We hope to see each colony glowing, with different brightnesses
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====Protocol====
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*Followed Jim Stock's protocol using his EX comp buffer
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*Left growing in 30°C incubator over a few days
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====Results====
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{| class="wikitable"
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|-
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! Strain
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! Plasmid Added
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! Glowing
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! Colonies
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|-
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| TOP10 with pHK555
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| pHK724
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| Yes
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| Yes
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|-
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| Red strain with pHK555
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| pHK724
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| No
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| Yes
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|-
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| Black strain with pHK555
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| pHK724
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| No
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| Yes
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|}
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====Conclusion====
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Only TOP10 successful however colonies seen on both of red and black strain plates.
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====Further Work====
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Plate out red and black strain colonies - possible that they had been left to incubate for too long and glowed then stopped glowing.
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Revision as of 10:51, 10 August 2010

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