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Team:Paris/Notebook/Protocols
From 2008.igem.org
Contents |
Culture of Stable strain with biobricks 2008
- In 6ml LB with adaptated antibiotics
- Will be use for Miniprep and Stock in glycerol
- 2 clones isolated by Biobricks
- O/N at 37°C
Glycerol Stocks
- 1mL of each culture (with 2 clones) has been added to 1mL of 40% glycerol.
- For each clone, two glycerol stocks have been done.
- Stored at -20°C.
Minipreps (Kit Qiagen)
- Resuspend pelleted bacterial cells in 250 µl Buffer P1 and transfer to a microcentrifuge tube.
- Add 250 µl Buffer P2 and mix thoroughly by inverting the tube 4–6 times. If using LyseBlue reagent, solution turns blue.
- Add 350 µl Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times. If using LyseBlue reagent, solution turns colorless.
- Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.
- Apply the supernatant (from step 4) to the QIAprep spin column by decanting or pipetting.
- Centrifuge for 30–60 s. Discard the flow-through.
- Recommended: Wash the QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30–60 s. Discard the flow-through. This step is only required when using endA+ or other bacteria strains with high nuclease activity or carbohydrate content (see QIAprep Miniprep Handbookfor more details)
- Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for 30–60 s.
- Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer.
- To elute DNA, place the QIAprep column in a clean 1.5 ml microcentrifuge tube. Add 50 µl Buffer EB or water to the center of each QIAprep spin column, let stand for 1 min, and centrifuge for 1 min.
Qualitative and quantitative analysis by electrophoresis
- Gel : 1% agarose
- 10 µL Quick-Load 1 kb DNA Ladder
- 2 µL LB + 3 µL DNA
=> Comparing the concentration of the miniprep thanks to the ladder
Check if the mesured size corresponds with the expecting size.
Concentration of the Miniprep
By biophotometry
- Blank : 60 µL of pure water
- Sample : 50 µL of pure water + 5 µLof DNA
Check if the ratio 260/280 is over 1,6
!!!Think about the dilution!!!
