Team:Paris/Notebook/Protocols

From 2008.igem.org

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==Migration after digestion==
==Migration after digestion==
 +
===Separation of insert and vector===
* Run the whole samples in a '''1,5% agarose gel'''  
* Run the whole samples in a '''1,5% agarose gel'''  
* About 30 minutes at 100 W   
* About 30 minutes at 100 W   

Revision as of 12:50, 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

Separation of insert and vector

  • 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


Purification

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