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Team:UCL Academy/Notebook/Week 3
From 2014hs.igem.org
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WEEK 3 | ||||||
Preparation of the DNA sample for CIDEB-UANIL’s team in Mexico
Aim: To prepare and deliver irrE DNA to the CIDEB-UANIL’s team.Tuesday 3rd June 2014.3 different glycerol stocks containing iRRE 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 4th June, 2014.Two LB tubes had grown overnight and these were spun at 5000RPM for 10 minutes. The pellets were frozen. All 3 agar plates had grown clear bacterial colonies. One was picked from each and added to 10 ml of LB medium. These were grown overnight at 37oC, 250RPM. Thursday 5th June, 2014.All three samples had grown and were spun at 5000RPM for 10 minutes. 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 with a nanodrop and the samples were sent with FedEx.
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