Team:Paris/Notebook/Protocols

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* 4 µl of screening PCR  
* 4 µl of screening PCR  
* migration ~30min at 100W on '''1,5%''' gel
* migration ~30min at 100W on '''1,5%''' gel
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==Sequencing==

Revision as of 14:12, 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 (a new one)
  • 10 µL of ladder 1 kb
  • whole 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)

Gel Slice and PCR Product Preparation

Dissolving the Gel Slice

  • Following electrophoresis, excise DNA band from gel slice in a 1.5 mL microcentrifuge tube.
  • Add 10µL membrane Binding Solution per 10 mg of gel slice. We prefer do not vortex and we incubate at 50-65°C until gel slice is completely dissolved (∼10 min). Centrifuge.

Processing PCR reactions

For products under 40 pb

  • Add an equal volume of Membrane Binding Solution to the PCR reaction.

Binding of DNA

  • Insert the SV Minicolumn into Collection Tube.
  • Transfer dissolved gel mixture or prepared PCR product to the Minicolumn assembly. Incubate at room temperature for 1 minute.
  • Centrifuge at 16,000 x "g for 1 minute. Discard the flowthrough and reinsert Minicolumn into Collection Tube.

Washing

  • Add 700 µL Membrane Wash Solution (ethanol added). Centrifuge at 16,000 x "g" for 1 minute. Discard flowthrough and reinsert the Minicolumn into Collection Tube.
  • Repeat Step 4 with 500 µL Membrane Wash Solution. Centrifuge at 16,000 x "g" for 5 minutes.
  • Empty the Collection Tube and recentrifuge the column assembly for 1 min with the microcentrifuge lid open (or off) to allow evaporation of any residual ethanol.

Elution

  • Carefully transfer Minicolumn to a clean 1.5 mL microcentrifuge tube.
  • Add 30 µL Buffer EB (Qiagen). Incubate at room temparature for 1 minute. Centifuge at 16,000 x "g" for 1 minute.
  • Discard Minicolumn and store at 4 or -20°C.


Ligation

  • 2 µL Ligase Buffer 10X
  • X µL insert
  • X/3 µL vector
  • Pure water qsp 20 µL
  • 1 µL T4 ligase
  • O/N at 16°C


Transformation

Use of TOP10 chemically competentcells

  • Defroze competent cells on ice during 5'
  • Add 5µl of DNA Ligation in 50µL of competent bacterias (or 1µL for the positive control puc19)
  • Incubate 30' on ice
  • Heat-shock the cells during 30" at 42°C without shaking
  • Put 2' on ice
  • Add 250µL of pre-warmed SOC medium (4°C)
  • Incubate 1h at 37°C under shaking (225rpm)
  • Spin at 5.000rpm during 30"
  • Remove 150µL of supernatant
  • Resuspent the pellet in the 150µL left
  • Spread on adequated plates
  • Incubate O/N at 37°C


Screening PCR

Use of 8 clones of Ligation transformants for screening PCR

  • one toothpick of each clone's colony by tube
  • In the same time cloning of the transformants (8 clones ; by streaks)

After, add

  • 25µL Mix
  • 1µL Oligo F (10µM)
  • 1µL Oligo R (10µM)
  • 23µL pure water
  • Program : SCREENIN

LID 105°C
1. 95°C 5min
2. 95°C 30 sec
3. 55°C 30 sec
4. 72°C 1 min 30 sec
5. go to : 2 rep : 29
6. sound : 1
7. hold : 4°C


Electrophoresis Purification of PCR

  • 10µl of ladder 100 pb or 1 kb
  • 4 µl of screening PCR
  • migration ~30min at 100W on 1,5% gel


Sequencing

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