Team:UCL Academy/Notebook/Week 4

From 2014hs.igem.org

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<h4>DNA concentrations were measured using a nanodrop, these were sufficiently high for PCR to be carried out.<h4/>
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<h4>DNA concentrations were measured using a nanodrop, these were sufficiently high for PCR to be carried out.</h4>
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Phusion polymerase PCR was carried out on our samples.
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<h4>PCR protocol used:</h4>
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We carried out transformation on competent cells in order to put together our composite biobrick.
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We transformed the constitutive promoter (part BBa_J23100), the repressor gene (part BBa_C0040), and the repressor regulated promoter (part BBa_R0040) into pre-bought competant cells.
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<br> 10microL of the resuspended biobrick was placed into 200microL of cells and left on ice. <br> <br>
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<h4>Our transformations were successful </h4>
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Latest revision as of 02:36, 21 June 2014

WEEK 4

Growing the bacteria needed for our own project

Aim: To grow two different bacterial strains for gene isolation

Tuesday 10th June, 2014.


WT E.coli, IrrE containing E. coli and Indoli glycerol stocks were streaked onto agar plates and grown overnight at 37oC. In addition, 10 microlitres of each sample was added to 10ml of LB media and grown overnight at 37oC, 250RPM.

Wednesday 11th June, 2014.


All the LB samples had grown and were spun at 5000RPM for 10 minutes. The pellets were frozen.
One colony per agar plate was picked and added to 10 ml of LB medium. These were grown overnight at 37oC, 250RPM.

Thursday 12th June, 2014.


Samples were spun at 5000RPM for 10 minutes. A mini-prep was carried out on these and the frozen pellets from the previous day.
A mini-prep was carried out on the pellets as well as those from the previous day.

Mini-prep protocol:.


1. Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to a microcentrifuge tube.
2. Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4–6 times.
3. Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times.
4. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.
5. Apply the supernatant from step 5 to the QIAprep spin column by decanting or pipetting.
6. Centrifuge for 30–60 s and discard the flow-through.
7. Wash the QIAprep spin column by adding 0.5 ml Buffer PB.
8. Wash the QIAprep spin column by adding 0.75 ml Buffer PE.
9. Centrifuge for 30–60 s and discard the flow-through
10. Centrifuge for 1 min to remove residual wash buffer
11. Place the QIAprep column in a clean 1.5 ml microcentrifuge tube.
12. Add 50 μl Buffer EB or water to the QIAprep spin column, stand for 1 min, and centrifuge for 1 min.

DNA concentrations were measured using a nanodrop, these were sufficiently high for PCR to be carried out.



Phusion polymerase PCR was carried out on our samples.

PCR protocol used:





We carried out transformation on competent cells in order to put together our composite biobrick. We transformed the constitutive promoter (part BBa_J23100), the repressor gene (part BBa_C0040), and the repressor regulated promoter (part BBa_R0040) into pre-bought competant cells.
10microL of the resuspended biobrick was placed into 200microL of cells and left on ice.

Our transformations were successful

.





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