Team:Paris/Notebook/Protocols

From 2008.igem.org

(Difference between revisions)
(→Migration after digestion)
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Check if the ratio 260/280 is over 1,6<br>!!!Think about the dilution!!!
Check if the ratio 260/280 is over 1,6<br>!!!Think about the dilution!!!
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==Amplification of promoters==
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=>To amplify the sequence in order to have enough amount of DNA to carry out the following of our experiments.''
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* '''Preparation of the templates''' : Resuspend of 1 colony  in 100µl of water.
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* '''Preparation of PCR mix''' :
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''For each samples,''
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 +
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1 µl dNTP
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<br>10 µl Buffer Phusion 5x
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<br>2,5 µl Oligo_F
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<br>2,5 µl Oligo_R
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<br>1µl template
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<br>1 µl Phusion
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<br>50 µl qsp H2O (33µl)
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*Program PCR : PROMOTEU
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LID : 105°C<br>
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1. 95°C  5 min<br>
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2. 95°C  1 min<br>
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3. 60°C  30 sec<br>
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4. 72°C  1 min 30 sec<br> (1 min for 1 kb)
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5. go to : 2  rep : 29
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6. sound : 1
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7. hold : 10°C   
==Digestion==
==Digestion==
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* 2 µL of LB
* 2 µL of LB
* Run at 50 W about 30 min
* Run at 50 W about 30 min
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==Extraction==
==Extraction==
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==Purification==
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==Purification (Kit Promega)==

Revision as of 13:07, 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!!!


Amplification of promoters

=>To amplify the sequence in order to have enough amount of DNA to carry out the following of our experiments.

  • Preparation of the templates : Resuspend of 1 colony in 100µl of water.
  • Preparation of PCR mix :

For each samples,


1 µl dNTP
10 µl Buffer Phusion 5x
2,5 µl Oligo_F
2,5 µl Oligo_R
1µl template
1 µl Phusion
50 µl qsp H2O (33µl)

  • Program PCR : PROMOTEU

LID : 105°C
1. 95°C 5 min
2. 95°C 1 min
3. 60°C 30 sec
4. 72°C 1 min 30 sec
(1 min for 1 kb) 5. go to : 2 rep : 29 6. sound : 1 7. hold : 10°C

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 (Kit Promega)

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