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Team:Paris/Notebook/Protocols
From 2008.igem.org
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!!!Separate each band by an empty one!!! | !!!Separate each band by an empty one!!! | ||
| + | ===For promoters=== | ||
| + | |||
| + | * 1,5% agarose gel | ||
| + | * 10 µL of ladder 100 pb | ||
| + | * 3 µL of digestion product | ||
| + | * 2 µL of LB | ||
| + | * Run at 100 W about 30 min | ||
| + | |||
| + | |||
| + | ===For vectors=== | ||
| + | |||
| + | * 1% agarose gel | ||
| + | * 10 µL of ladder 1 kb | ||
| + | * 3 µL of digestion product | ||
| + | * 2 µL of LB | ||
| + | * Run at 50 W about 30 min | ||
==Extraction== | ==Extraction== | ||
Revision as of 12:48, 11 August 2008
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
- Bath of BET 20000X (5 µL BET for 100 mL TBE) during about 5 min.
=> 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!!!
Digestion
- 1 µg of plasmid (Miniprep)
- Buffer (n°2) 10X
- BSA 100X
- Pure water qsp 30 µL
- 1 µL of each enzyme
- Incubate during about 3h at 37°C, then 10 minutes at 60-65°C (to inactivate the enzymes).
Migration after digestion
- Run the whole samples in a 1,5% agarose gel
- About 30 minutes at 100 W
- 10 µL of ladder 1kb and 100 pb on every side
- 3 µL of DNA + 2 µL of LB
!!!Separate each band by an empty one!!!
For promoters
- 1,5% agarose gel
- 10 µL of ladder 100 pb
- 3 µL of digestion product
- 2 µL of LB
- Run at 100 W about 30 min
For vectors
- 1% agarose gel
- 10 µL of ladder 1 kb
- 3 µL of digestion product
- 2 µL of LB
- Run at 50 W about 30 min
Extraction
- For each new extraction it's important to have a new bath of BET
- Use a new blade for each extraction
- The extraction must be under 400 mg
