Team:Paris/Notebook/Protocols

From 2008.igem.org

(Difference between revisions)
(Transformation)
(PCR Screening)
 
(44 intermediate revisions not shown)
Line 5: Line 5:
* Streaks on plates with LB and the adapted antibiotics to isolate colonies
* Streaks on plates with LB and the adapted antibiotics to isolate colonies
* Incubate O/N at 37°C
* Incubate O/N at 37°C
-
* Take clone with a toothpick and put in 7.5ml LB with adaptated antibiotics (LB have to be prepared before !!!)
+
* Take clone with a sterile tip and put in 7.5ml LB with adaptated antibiotics (LB have to be prepared before !!!)
* Will be use for Miniprep and Stock in glycerol
* Will be use for Miniprep and Stock in glycerol
* 2-3 clones isolated by Biobricks
* 2-3 clones isolated by Biobricks
Line 28: Line 28:
* Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for 30–60 s.  
* 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.  
* 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 30 µl Buffer EB or water to the center of each QIAprep spin column, let stand for 1 min, and centrifuge for 1 min.
+
* To elute DNA, place the QIAprep column in a clean 1.5 ml microcentrifuge tube. Add 30 µl Buffer EB or pure water to the center of each QIAprep spin column, let stand for 1 min, and centrifuge for 1 min.
= Electrophoresis =
= Electrophoresis =
Line 34: Line 34:
* Gel : 1% agarose with BET added (5 µL BET for 100 mL TBE)
* Gel : 1% agarose with BET added (5 µL BET for 100 mL TBE)
* 10 µL Quick-Load 1 kb DNA Ladder
* 10 µL Quick-Load 1 kb DNA Ladder
-
* 2 µL LB + 3 µL DNA
+
* 2 µL Loading Dye + 3 µL DNA
-
+
-
= Concentration of the Miniprep =
+
= Concentration of the Miniprep or the Midiprep =
By biophotometry
By biophotometry
-
* Blank : 55 µL of pure water + 5 µL EB
+
* Blank : 60 µL of pure water  or 55 µL of pure water + 5 µL EB
-
* Sample : 55 µL of pure water + 5 µL of DNA
+
* Sample : 55 µL of pure water + 5 µL of sample
Check if the ratio 260/280 is over 1,6
Check if the ratio 260/280 is over 1,6
'''Think about the dilution !'''
'''Think about the dilution !'''
 +
 +
If the concentration is to low, concentrate the sample in a vacuum centrifuge. Don't forget to open the tubes!
= Digestion =
= Digestion =
-
* 1 µg of plasmid / 250 ng of gene
+
* 1 µg of plasmid / 250 ng of PCR product
* Buffer (n°2) 10X : 3µL
* Buffer (n°2) 10X : 3µL
* BSA 100X : 0.3µL
* BSA 100X : 0.3µL
-
* Pure water qsp 30 µL
 
* 1 µL of each enzyme
* 1 µL of each enzyme
 +
* Pure water qsp 30 µL
* Incubate during about 2h30-3h at 37°C, then 20 minutes at 65°C (to inactivate the enzymes).
* Incubate during about 2h30-3h at 37°C, then 20 minutes at 65°C (to inactivate the enzymes).
= Migration after digestion =
= Migration after digestion =
-
* Run the whole samples (30 µL) in a '''0.8-1% agarose gel''' (a new one)
+
* Run the whole samples (30 µL) in a '''1-2% agarose gel with TAE buffer''' (a new one)  
* Run at 50 V until halfway   
* Run at 50 V until halfway   
* 10 µL of ladder 1kb and 100 pb on every side
* 10 µL of ladder 1kb and 100 pb on every side
-
* 30 µL of DNA + 6 µL of LB
+
* 30 µL of DNA + 6 µL of Loading Dye
-
'''Separate each band by an empty one !'''
+
'''Separate each sample by an empty well !'''
= Extraction =
= Extraction =
-
* For each new extraction it's important to have a new bath of ETB
+
* For each new extraction it's important to have a new bath of TAE Buffer for the run and of ETB for the revelation
 +
* Use UV at lower intensity and for shorter time as possible
* Use a new blade for each extraction
* Use a new blade for each extraction
-
* The band weight must be less than 200 mg
+
* The band weight must be less than 400 mg
 +
 
 +
 
 +
= Antarctic phosphatase =
 +
To avoid autoligation, the vector in a construction has to be treated with phosphatase to dephosphorylate the cut end of the plasmid. The Antarctic phosphatase has the advantage to be easily and totally inactivated by heating.
 +
* After the digestion reaction add Antarctic phosphatase buffer to a final concentration of 1x
 +
* Add 1µL of Antarctic phosphatase for 1 to 5 µg of DNA
 +
* Incubate at 37°C for 15 minutes and then inactivate the phosphatase at 65°C for 5 min
 +
 
 +
 
 +
'''Do not use this reaction for the insert or the ligation will not work'''
= Amplification of promoters =
= Amplification of promoters =
Line 94: Line 106:
8. hold : 10°C
8. hold : 10°C
-
= Purification (PROMEGA kit) =
+
= Purification (PROMEGA or QIAcube or QIAquick kit) =
== Gel Slice and PCR Product Preparation ==
== Gel Slice and PCR Product Preparation ==
=== Dissolving the Gel Slice ===
=== Dissolving the Gel Slice ===
-
* Following electrophoresis, excise DNA band from gel slice in a pre-weighed 1.5 mL microcentrifuge tube.
+
* Following electrophoresis, excise DNA band from gel slice in a pre-weighed 2 mL microcentrifuge tube.
* Add 10µL membrane Binding Solution per 10 mg of gel slice. We prefer not to vortex and we incubate at 50-65°C until gel slice is completely dissolved (∼10 min). Quick centrifuge.
* Add 10µL membrane Binding Solution per 10 mg of gel slice. We prefer not to vortex and we incubate at 50-65°C until gel slice is completely dissolved (∼10 min). Quick centrifuge.
Line 117: Line 129:
== Elution ==
== Elution ==
* Carefully transfer Minicolumn to a clean 1.5 mL microcentrifuge  tube.
* 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.
+
* Add 30 µL of pure water or 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.
* Discard Minicolumn and store at 4 or -20°C.
-
 
= Quantification by Electrophoresis =
= Quantification by Electrophoresis =
* Gel : 1.5-2% agarose with BET added (5 µL BET for 100 mL TBE)
* Gel : 1.5-2% agarose with BET added (5 µL BET for 100 mL TBE)
-
* 10 µL Quick-Load 1 kb DNA Ladder
+
* 10 µL Quick-Load 100pb or 1 kb DNA Ladder
-
* 2 µL LB + 3 µL DNA
+
* 2 µL Loadind Dye + 3 µL DNA
-
 
+
= Ligation =
= Ligation =
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* Spread on appropriate plates
* Spread on appropriate plates
* Incubate O/N at 37°C
* Incubate O/N at 37°C
 +
 +
 +
= LB and LBA =
 +
 +
* LB medium: Dissolve 25mg/L of Luria Broth in purified water. Autoclave the solution. Store at room temperature and take care of any contamination
 +
 +
* LBA medium: Dissolve and gently mix with a magnetic barrel 40mg/L of Luria Broth Agar in purified water. Autoclave the solution. Store at room temperature and take care of any contamination. To make some plates heat the solidified medium with microwave. Wait untill you can hold the bottle in your hands and then add the appropriate antibiotic to a final concentration of 1x. Provide approximatively 20mL per plate in a sterile hood. Returned the plates and store them at +4°C
 +
 +
= Minimum Medium =
 +
 +
*Minimum Medium:
 +
- B1 Vitamin 0.1%
 +
<br>- Uracile 0.2%
 +
<br>- MgSO<sub>4</sub> 2 mM
 +
<br>- CaCl<sub>2</sub> 0.1 mM
 +
<br>- Glycerol 0.4%
 +
<br>- Casamino acids 0.1%
 +
<br>- M9 minimal salts medium 1x
 +
<br>- H<sub>2</sub>O QSP 200 mL
 +
 +
Filter the medium with a cell-culture unit of filtration.  If needed add the appropriate antibiotic to a final concentration of 1x
 +
 +
= Soft Agar Medium =
 +
 +
* Add 3.5 to 4g of agar per liter of LB or Minimum Medium. Autoclave the solution. Store at room temperature and take care of any contamination. To make some plates heat the solidified medium with microwave. Wait untill you can hold the bottle in your hands and then add the appropriate antibiotic to a final concentration of 1x. Provide approximatively 20mL per plate in a sterile hood. Returned the plates and store them at +4°C
 +
 +
= 1000x stock antibiotic =
 +
 +
* Chloramphenicol: dissolve 30mg/mL of chloramphenicol in ethanol 100%. Store at room temperature
 +
* Tetracyclin: dissolve 12,5mg/mL of tetracyclin in a 50/50 ethanol/water mix. Store at -20°C
 +
* Ampicillin: dissolve 100mg/mL of ampicillin in water. Filter the solution. Store at +4°C
 +
* Kanamycin: dissolve 100mg/mL of kanamycin in water. Filter the solution. Store at room temperature
= PCR Screening =
= PCR Screening =
Use of 8 clones of Ligation transformants for screening PCR
Use of 8 clones of Ligation transformants for screening PCR
-
* One toothpick of each clone's colony per PCR tube
+
* One sterile tip of each clone's colony per PCR tube
-
* Use toothpick to start 7.5mL O/N culture
+
* Use sterile tip to start 7.5mL O/N culture
'''After''', add
'''After''', add
* 12.5 µL Mix
* 12.5 µL Mix
-
* 0.5 µL Oligo F (10µM)
+
* 0.5 µL Oligo VF (10µM)
-
* 0.5 µL Oligo R (10µM)
+
* 0.5 µL Oligo VR (10µM)
* 11,5 µL pure water
* 11,5 µL pure water
*negative control : without clone's colony
*negative control : without clone's colony
Line 179: Line 221:
6. sound : 1<br>
6. sound : 1<br>
7. hold : 4°C
7. hold : 4°C
-
 
= Electrophoresis Purification of PCR =
= Electrophoresis Purification of PCR =
Line 232: Line 273:
|yes
|yes
|- style="text-align: center;"
|- style="text-align: center;"
-
|PflhDC
+
|pFlhDC
-
|?
+
|yes
|- style="text-align: center;"
|- style="text-align: center;"
-
|PfliA
+
|pFliA
-
|?
+
|yes
|- style="text-align: center;"
|- style="text-align: center;"
-
|PfliL
+
|pFliL
-
|?
+
|yes
|- style="text-align: center;"
|- style="text-align: center;"
-
|PflgA
+
|pFlgA
|yes
|yes
|- style="text-align: center;"
|- style="text-align: center;"
-
|PflgB
+
|pFlgB
|yes
|yes
|- style="text-align: center;"
|- style="text-align: center;"
-
|PflhB
+
|pFlhB
|yes
|yes
|- style="text-align: center;"
|- style="text-align: center;"
Line 255: Line 296:
|yes
|yes
|- style="text-align: center;"
|- style="text-align: center;"
-
|gfp E0240
+
|GFP E0240
|yes
|yes
|- style="text-align: center;"
|- style="text-align: center;"
-
|ompR*
+
|OmpR*
-
|?
+
|no
|- style="text-align: center;"
|- style="text-align: center;"
-
|envZ*
+
|EnvZ*
-
|?
+
|yes
|- style="text-align: center;"
|- style="text-align: center;"
-
|fliA
+
|FliA
-
|no
+
|yes
|- style="text-align: center;"
|- style="text-align: center;"
-
|flhDC
+
|FlhDC
|yes
|yes
|- style="text-align: center;"
|- style="text-align: center;"
|colspan="4"|<center>'''Plasmids'''</center>
|colspan="4"|<center>'''Plasmids'''</center>
|- style="text-align: center;"
|- style="text-align: center;"
-
|PSB3K3
+
|pSB3K3 (ORI p15A)
 +
|yes
 +
|- style="text-align: center;"
 +
|pSB4T5 (ORI pSC101)
|yes
|yes
|- style="text-align: center;"
|- style="text-align: center;"
Line 278: Line 322:
|- style="text-align: center;"
|- style="text-align: center;"
|B0032
|B0032
-
|?
+
|yes
|- style="text-align: center;"
|- style="text-align: center;"
|colspan="4"|<center>'''Terminators'''</center>
|colspan="4"|<center>'''Terminators'''</center>
|- style="text-align: center;"
|- style="text-align: center;"
|B0010
|B0010
-
|?
+
|yes
|- style="text-align: center;"
|- style="text-align: center;"
|B0012
|B0012
-
|?
+
|yes
|- style="text-align: center;"
|- style="text-align: center;"
|colspan="4"|<center>'''Bacterial Strains'''</center>
|colspan="4"|<center>'''Bacterial Strains'''</center>
Line 294: Line 338:
|- style="text-align: center;"
|- style="text-align: center;"
|FliA -/ FlhDC -
|FliA -/ FlhDC -
-
|ask Ariel
+
|yes
|- style="text-align: center;"
|- style="text-align: center;"
|FlgM -
|FlgM -
-
|ask Ariel
+
|yes
|}
|}
Line 326: Line 370:
|yes
|yes
|
|
-
|?
+
|
|
|
|- style="text-align: center;"
|- style="text-align: center;"
Line 335: Line 379:
|
|
|- style="text-align: center;"
|- style="text-align: center;"
-
|None
+
|
-
|?
+
|
-
|gfp E0240
+
|GFP E0240
|yes
|yes
|
|
|- style="text-align: center;"
|- style="text-align: center;"
-
|PflhDC
+
|pFlhDC
-
|?
+
|yes
-
|ompR*
+
|OmpR*
-
|?
+
|no
|
|
|- style="text-align: center;"
|- style="text-align: center;"
-
|PflhDC
+
|pFlhDC
-
|?
+
|yes
-
|envZ*
+
|EnvZ*
-
|?
+
|yes
|
|
|- style="text-align: center;"
|- style="text-align: center;"
-
|PflhDC
+
|pFlhDC
-
|?
+
|yes
-
|fliA
+
|FliA
-
|no
+
|yes
|
|
|- style="text-align: center;"
|- style="text-align: center;"
-
|PfliA
+
|pFliA
-
|?
+
|yes
-
|flhDC
+
|FlhDC
|yes
|yes
|
|
|- style="text-align: center;"
|- style="text-align: center;"
-
|PfliA
+
|pFliA
-
|?
+
|yes
-
|fliA
+
|FliA
-
|no
+
|yes
|
|
|- style="text-align: center;"
|- style="text-align: center;"
-
|PfliL
+
|pFliL
-
|?
+
|yes
-
|flhDC
+
|FlhDC
|yes
|yes
|
|
|- style="text-align: center;"
|- style="text-align: center;"
-
|PfliL
+
|pFliL
-
|?
+
|yes
-
|fliA
+
|FliA
-
|no
+
|yes
|
|
|- style="text-align: center;"
|- style="text-align: center;"
-
|PflgA
+
|pFlgA
|yes
|yes
-
|flhDC
+
|FlhDC
|yes
|yes
|
|
|- style="text-align: center;"
|- style="text-align: center;"
-
|PflgA
+
|pFlgA
 +
|yes
 +
|FliA
|yes
|yes
-
|fliA
 
-
|no
 
|
|
|- style="text-align: center;"
|- style="text-align: center;"
-
|PflgB
+
|pflgB
|yes
|yes
-
|flhDC
+
|FlhDC
|yes
|yes
|
|
|- style="text-align: center;"
|- style="text-align: center;"
-
|PflgB
+
|pFlgB
 +
|yes
 +
|FliA
|yes
|yes
-
|fliA
 
-
|no
 
|
|
|- style="text-align: center;"
|- style="text-align: center;"
-
|PflhB
+
|pFlhB
|yes
|yes
-
|flhDC
+
|FlhDC
|yes
|yes
|
|
|- style="text-align: center;"
|- style="text-align: center;"
-
|PflhB
+
|pFlhB
 +
|yes
 +
|FliA
|yes
|yes
-
|fliA
 
-
|no
 
|
|
|}
|}
Line 422: Line 466:
= Protocol to make competent bacteria =
= Protocol to make competent bacteria =
-
1. Use non competent bacteria (ex: MG1655) stocked in 1.5 mL LB (20% Glycerol): put a toothpick in the 1.5 mL stock tube and then place it in a 50 mL Falcon with 5 ml LB medium. Over Night culture at 37°C / 300 rpm
+
1. Use non competent bacteria (ex: MG1655) stocked in 1.5 mL LB (20% Glycerol): put a sterile tip in the 1.5 mL stock tube and then place it in a 50 mL Falcon with 5 ml LB medium. Over Night culture at 37°C / 300 rpm
2. 1/100 dilution in LB medium QSP 50 mL in an erlenmeyer of 250 mL
2. 1/100 dilution in LB medium QSP 50 mL in an erlenmeyer of 250 mL
Line 449: Line 493:
9. Discard supernatant by inverting the tube, and resuspend the pellet with 1 mL of cold CaCl<sub>2</sub> and mix gently the suspension by up and down
9. Discard supernatant by inverting the tube, and resuspend the pellet with 1 mL of cold CaCl<sub>2</sub> and mix gently the suspension by up and down
-
10. Pre-warm the bain-marie at 42°C
+
10. [https://2008.igem.org/Team:Paris/Notebook/Protocols#Transformation Transform] or freeze the competent cells. Freeze the competent cells in 50 µL aliquots in the 0.1M CaCl<sub>2</sub> medium with 15% glycerol.
-
11. Add 2 µL of 1/100 diluted plasmid (containing a strong constitutive promoter [http://partsregistry.org/Part:BBa_J23101 J23101] before the [http://partsregistry.org/Part:BBa_E0240 GFP tripart]) to 100 µL of competent bacteria in a 15 ml falcon
+
11. After transformation, prepare a [https://2008.igem.org/Team:Paris/Notebook/Protocols#Glycerol_Stocks Glycerol Stock] or/and use the transformed bacteria to study [https://2008.igem.org/Team:Paris/Notebook/Protocols#Study_of_the_doubling_time_of_the_bacteria_population the doubling time of the bacteria population]
-
 
+
-
12. Incubate 20 min at +4°C
+
-
 
+
-
13. Heat-Shock: 40 sec at 42°C / then 1 min in ice
+
-
 
+
-
14. Add to the sample 1 mL LB medium without antibiotic and incubate 1h at 37°C / 300 rpm
+
-
 
+
-
15. Spread 10 to 100 µL on plates with LB medium and the appropriate antibiotic
+
-
 
+
-
16. Incubate O/N at 37°C / 300 rpm
+
-
 
+
-
17. Prepare a [https://2008.igem.org/Team:Paris/Notebook/Protocols#Glycerol_Stocks Glycerol Stock] or/and use the transformed bacteria to study [https://2008.igem.org/Team:Paris/Notebook/Protocols#Study_of_the_doubling_time_of_the_bacteria_population the doubling time of the bacteria population]
+
= Study of the doubling time of the bacteria population =
= Study of the doubling time of the bacteria population =
-
*Medium:
 
-
- B1 Vitamin 0.1%
 
-
<br>- Uracile 0.2%
 
-
<br>- MgSO<sub>4</sub> 2 mM
 
-
<br>- CaCl<sub>2</sub> 0.1 mM
 
-
<br>- Glycerol 0.4%
 
-
<br>- Casamino acids 0.1%
 
-
<br>- M9 minimal salts medium 1x
 
-
<br>- H<sub>2</sub>O QSP 200 mL
 
-
* Filter the medium with a cell-culture unit of filtration
 
* Unfreeze strains from the glycerol stock containing or not the plasmid with a contitutive promoter and a fluorescent reporter gene
* Unfreeze strains from the glycerol stock containing or not the plasmid with a contitutive promoter and a fluorescent reporter gene
-
* Put a toothpick in the 1.5 mL stock tube and then place it in a 50 mL Falcon with 5 ml of the medium above. Over Night (16h) culture at 37°C / 300 rpm
+
* Put a sterile tip in the 1.5 mL stock tube and then place it in a 50 mL Falcon with 5 ml of [https://2008.igem.org/Team:Paris/Notebook/Protocols#Minimum_Medium minimum medium]. Over Night (16h) culture at 37°C / 300 rpm
* Measure the OD<sub>600</sub> (Linear Zone Measurement <1.5)
* Measure the OD<sub>600</sub> (Linear Zone Measurement <1.5)
* Dilute the culture medium in the same new medium to obtain an approximative OD<sub>600</sub> of 0.01 (V<sub>final</sub>= 50 mL in a 250 ml erlenmeyer)
* Dilute the culture medium in the same new medium to obtain an approximative OD<sub>600</sub> of 0.01 (V<sub>final</sub>= 50 mL in a 250 ml erlenmeyer)
* Incubate à 37°C / 300 rpm and measure the OD<sub>600</sub> every 20 min
* Incubate à 37°C / 300 rpm and measure the OD<sub>600</sub> every 20 min
* Determine the doubling time population and compare the strains containing or not the plasmid with a contitutive promoter and a fluorescent reporter gene
* Determine the doubling time population and compare the strains containing or not the plasmid with a contitutive promoter and a fluorescent reporter gene

Latest revision as of 12:45, 21 October 2008

Contents

Culture of Stable strain with biobricks 2008

  • Streaks on plates with LB and the adapted antibiotics to isolate colonies
  • Incubate O/N at 37°C
  • Take clone with a sterile tip and put in 7.5ml LB with adaptated antibiotics (LB have to be prepared before !!!)
  • Will be use for Miniprep and Stock in glycerol
  • 2-3 clones isolated by Biobricks
  • Incubate O/N at 37°C (open just a little the cap for bacteria's breathing)
  • Don't store LB in the fridge even with antibiotics !

Glycerol Stocks

  • Remove 2.5mL of each culture and centrifuge.
  • Discard the supernatant and resuspend pelleted bacteria in 1mL of LB.
  • Add 500µL of 60% glycerol.
  • Store at -20°C.

Minipreps (QIAGEN kit)

  • centrifuge 5mL of culture 8 min at 3,500 to 4,000 g.
  • 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 30 µl Buffer EB or pure water to the center of each QIAprep spin column, let stand for 1 min, and centrifuge for 1 min.

Electrophoresis

An electrophoresis can be done to check if there is Product of Miniprep

  • Gel : 1% agarose with BET added (5 µL BET for 100 mL TBE)
  • 10 µL Quick-Load 1 kb DNA Ladder
  • 2 µL Loading Dye + 3 µL DNA

Concentration of the Miniprep or the Midiprep

By biophotometry

  • Blank : 60 µL of pure water or 55 µL of pure water + 5 µL EB
  • Sample : 55 µL of pure water + 5 µL of sample

Check if the ratio 260/280 is over 1,6

Think about the dilution !

If the concentration is to low, concentrate the sample in a vacuum centrifuge. Don't forget to open the tubes!

Digestion

  • 1 µg of plasmid / 250 ng of PCR product
  • Buffer (n°2) 10X : 3µL
  • BSA 100X : 0.3µL
  • 1 µL of each enzyme
  • Pure water qsp 30 µL
  • Incubate during about 2h30-3h at 37°C, then 20 minutes at 65°C (to inactivate the enzymes).

Migration after digestion

  • Run the whole samples (30 µL) in a 1-2% agarose gel with TAE buffer (a new one)
  • Run at 50 V until halfway
  • 10 µL of ladder 1kb and 100 pb on every side
  • 30 µL of DNA + 6 µL of Loading Dye

Separate each sample by an empty well !

Extraction

  • For each new extraction it's important to have a new bath of TAE Buffer for the run and of ETB for the revelation
  • Use UV at lower intensity and for shorter time as possible
  • Use a new blade for each extraction
  • The band weight must be less than 400 mg


Antarctic phosphatase

To avoid autoligation, the vector in a construction has to be treated with phosphatase to dephosphorylate the cut end of the plasmid. The Antarctic phosphatase has the advantage to be easily and totally inactivated by heating.

  • After the digestion reaction add Antarctic phosphatase buffer to a final concentration of 1x
  • Add 1µL of Antarctic phosphatase for 1 to 5 µg of DNA
  • Incubate at 37°C for 15 minutes and then inactivate the phosphatase at 65°C for 5 min


Do not use this reaction for the insert or the ligation will not work

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)

  • make a mix with buffer, oligos and water for n+1 samples
  • negative control : without template
  • positive control : known template
  • Program PCR : PHUSION2

LID : 105°C
1. 98°C 30 sec initial denaturation
2. 98°C 10 sec denaturation
3. 60°C (depending of the size of oligos) 30 sec annealing
4. 72°C (1 min for 1 kb)
5. go to : 2 rep : 24-29
6. 72°C 5 min
7. sound : 1
8. hold : 10°C

Purification (PROMEGA or QIAcube or QIAquick kit)

Gel Slice and PCR Product Preparation

Dissolving the Gel Slice

  • Following electrophoresis, excise DNA band from gel slice in a pre-weighed 2 mL microcentrifuge tube.
  • Add 10µL membrane Binding Solution per 10 mg of gel slice. We prefer not to vortex and we incubate at 50-65°C until gel slice is completely dissolved (∼10 min). Quick centrifuge.

Processing PCR reactions

For products above 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 of pure water or 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.

Quantification by Electrophoresis

  • Gel : 1.5-2% agarose with BET added (5 µL BET for 100 mL TBE)
  • 10 µL Quick-Load 100pb or 1 kb DNA Ladder
  • 2 µL Loadind Dye + 3 µL DNA

Ligation

  • 2 µL Ligase Buffer 10X
  • Vv µL of measured [DNA]Vector in ng/µL (30 to 50 ng)
  • Vi µL of measured [DNA]Insert in ng/µL
  • Pure water QSP 20 µL
  • 1 µL T4 DNA ligase at 400 000 U/mL concentration
  • O/N at 16°C


Vi = (3 x [DNA]Vector x Vv x LenghtInsert) / ([DNA]Insert x LenghtVector)

Transformation

Use chemically lab-made competent cells or from a kit (DH5α, TOP10, Mach1)

  • Defroze competent cells on ice during 5'
  • Add 5 µl of DNA Ligation in 50 µL of competent bacterias (or 1 µL 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 250 µL of pre-warmed SOC medium (42°C)
  • Incubate 1h at 37°C under shaking (225rpm)
  • Spin at 5.000rpm during 30"
  • Remove 150 µL of supernatant
  • Resuspend the pellet in the 150 µL left
  • Spread on appropriate plates
  • Incubate O/N at 37°C


LB and LBA

  • LB medium: Dissolve 25mg/L of Luria Broth in purified water. Autoclave the solution. Store at room temperature and take care of any contamination
  • LBA medium: Dissolve and gently mix with a magnetic barrel 40mg/L of Luria Broth Agar in purified water. Autoclave the solution. Store at room temperature and take care of any contamination. To make some plates heat the solidified medium with microwave. Wait untill you can hold the bottle in your hands and then add the appropriate antibiotic to a final concentration of 1x. Provide approximatively 20mL per plate in a sterile hood. Returned the plates and store them at +4°C

Minimum Medium

  • Minimum Medium:

- B1 Vitamin 0.1%
- Uracile 0.2%
- MgSO4 2 mM
- CaCl2 0.1 mM
- Glycerol 0.4%
- Casamino acids 0.1%
- M9 minimal salts medium 1x
- H2O QSP 200 mL

Filter the medium with a cell-culture unit of filtration. If needed add the appropriate antibiotic to a final concentration of 1x

Soft Agar Medium

  • Add 3.5 to 4g of agar per liter of LB or Minimum Medium. Autoclave the solution. Store at room temperature and take care of any contamination. To make some plates heat the solidified medium with microwave. Wait untill you can hold the bottle in your hands and then add the appropriate antibiotic to a final concentration of 1x. Provide approximatively 20mL per plate in a sterile hood. Returned the plates and store them at +4°C

1000x stock antibiotic

  • Chloramphenicol: dissolve 30mg/mL of chloramphenicol in ethanol 100%. Store at room temperature
  • Tetracyclin: dissolve 12,5mg/mL of tetracyclin in a 50/50 ethanol/water mix. Store at -20°C
  • Ampicillin: dissolve 100mg/mL of ampicillin in water. Filter the solution. Store at +4°C
  • Kanamycin: dissolve 100mg/mL of kanamycin in water. Filter the solution. Store at room temperature

PCR Screening

Use of 8 clones of Ligation transformants for screening PCR

  • One sterile tip of each clone's colony per PCR tube
  • Use sterile tip to start 7.5mL O/N culture

After, add

  • 12.5 µL Mix
  • 0.5 µL Oligo VF (10µM)
  • 0.5 µL Oligo VR (10µM)
  • 11,5 µL pure water
  • negative control : without clone's colony
  • positive control

Store the tubes on ice waiting for PCR attains 95°C then put the tubes in the machine

  • Program : SCREENIN

LID 105°C
1. 95°C 5min
2. 95°C 30 sec
3. 55°C 30 sec
4. 72°C (1 min for 1kb)
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 at 100V on 1,5% gel until 3/4 way

Sequencing

[http://institut.cochin.inserm.fr/rubric_recherche/Plates-Formes/sequencage_genomique/I18NFolder.2005-02-10.4781618697/page2/fr Sequencing COCHIN]



Promoter Characterization Plan

For theoretical consideration, see estimation of parameters

  • Standart construct

New work plan.png


  • Order of work

NW-Steps-3rd option.png NW-Steps-2nd option.png

Plan characterization-ptet charac.png

Plan characterization-pconst charac.png

The same colour coded steps can be perfomed at the same time if elements needed are available The order for treating the colours should of course be:

Colour coding for characterization.png


  • Material needed
Availability
Promoters
J23101 yes
pTet yes
pFlhDC yes
pFliA yes
pFliL yes
pFlgA yes
pFlgB yes
pFlhB yes
Genes
tetR yes
GFP E0240 yes
OmpR* no
EnvZ* yes
FliA yes
FlhDC yes
Plasmids
pSB3K3 (ORI p15A) yes
pSB4T5 (ORI pSC101) yes
RBS
B0032 yes
Terminators
B0010 yes
B0012 yes
Bacterial Strains
Top10 yes
FliA -/ FlhDC - yes
FlgM - yes



  • Summary table

This table contains the promoters we need to characterize, the transcription factors whose effect on the promoter we want to test, and the plasmid we want to obtain in order to carry out each characterization

Promoter Promoter

Availability

TF gene TF gene

Availability

Done/Not done
J23101 yes
pTet yes
tetR yes
GFP E0240 yes
pFlhDC yes OmpR* no
pFlhDC yes EnvZ* yes
pFlhDC yes FliA yes
pFliA yes FlhDC yes
pFliA yes FliA yes
pFliL yes FlhDC yes
pFliL yes FliA yes
pFlgA yes FlhDC yes
pFlgA yes FliA yes
pflgB yes FlhDC yes
pFlgB yes FliA yes
pFlhB yes FlhDC yes
pFlhB yes FliA yes

Protocol to make competent bacteria

1. Use non competent bacteria (ex: MG1655) stocked in 1.5 mL LB (20% Glycerol): put a sterile tip in the 1.5 mL stock tube and then place it in a 50 mL Falcon with 5 ml LB medium. Over Night culture at 37°C / 300 rpm

2. 1/100 dilution in LB medium QSP 50 mL in an erlenmeyer of 250 mL

3. Culture at 37°C / 300 rpm untill OD600 reach 0.6

4. Fast cooling at +4°C by gently shaking the erlen in ice


Before: prepare CaCl2 0.1M.

  • Add 5,56 g in 500 mL H2O (Gibco)
  • dissolve the CaCl2 by mixing the suspension with the help of a magnetic stirrer
  • Filter the solution with a cell-culture unit of filtration
  • Aliquotes the filtered solution: 25mL in a 50 mL Falcon. Storage at +4°C

5. Use pre-cooled centrifuge at +4°C. Centrifuge 50 mL of the culture in 50 mL falcon: +4°C / 5 min / 5000 rpm

6. Discard supernatant by inverting the tube, and resuspend the pellet with 1 mL of cold CaCl2 and mix gently the suspension by up and down

7. Add cold CaCl2 QSP 20 mL and incubate 30 min / +4°C

8. Centrifuge the suspension : +4°C / 5 min / 5000 rpm

9. Discard supernatant by inverting the tube, and resuspend the pellet with 1 mL of cold CaCl2 and mix gently the suspension by up and down

10. Transform or freeze the competent cells. Freeze the competent cells in 50 µL aliquots in the 0.1M CaCl2 medium with 15% glycerol.

11. After transformation, prepare a Glycerol Stock or/and use the transformed bacteria to study the doubling time of the bacteria population

Study of the doubling time of the bacteria population

  • Unfreeze strains from the glycerol stock containing or not the plasmid with a contitutive promoter and a fluorescent reporter gene
  • Put a sterile tip in the 1.5 mL stock tube and then place it in a 50 mL Falcon with 5 ml of minimum medium. Over Night (16h) culture at 37°C / 300 rpm
  • Measure the OD600 (Linear Zone Measurement <1.5)
  • Dilute the culture medium in the same new medium to obtain an approximative OD600 of 0.01 (Vfinal= 50 mL in a 250 ml erlenmeyer)
  • Incubate à 37°C / 300 rpm and measure the OD600 every 20 min
  • Determine the doubling time population and compare the strains containing or not the plasmid with a contitutive promoter and a fluorescent reporter gene