Paris/PROTOCOLS

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Latest revision as of 09:48, 4 September 2007

For newcomers in wetlab: see also the course by D. Endy [http://openwetware.org/wiki/20.109%28S07%29:Lab_tourlink Laboratory Fundamentals of Biological Engineering].

1 Getting started
2 Strains
3 Transduction with P1 bacteriophage
4 Preparation of DAP solution from the powder (50mM)
5 Preparing growth media
6 Chemical transformation
7 Glycerol Stock
8 Recombineering/Lambda red-mediated gene replacement
9 Miniprep
10 Fluorescent single cells visualisation
11 Digestion reactions
12 Preparation de gel d'agarose

Getting started

This topic is adressed to all our informatics-physics-I'm-afraid-of-the-bench fellows. So, if you finally found the courage to dare the pipettes, PCRs and nicely smelling bacteria, welcome!

What a pipette is?

Pipettes dispense various volumes. The plunger button indicates the maximum volume (microliters) that the pipette is designed to handle. For example, P-20 will handle up to 20 microliters.
The digital volume indicator is read from top to bottom. For P-2, P-10, P-20, P-100, and P-200, black digits indicate microliters and red digits tenths and hundredths of microliters. For P-1000, red digits indicate milliliters and black digits microliters.

What to do when you have it in you hand?

-Hold the pipette in one hand (it doesn't bite...). With the other hand, turn the volume adjustment knob counterclockwise so the volume indicator is 1/3 revolution above the desired setting, then slowly turn clockwise until the indicator shows the desired volume.
-Attach a new disposable tip to the pipette shaft.
-Press the plunger to the FIRST stop. This part of the stroke is the volume displayed by the indicator.
-Holding the pipette vertically, immerse the tip a few millimeters into the sample.
-Allow the pushbutton to return slowly to the UP position. Avoid to blurt out the plunger button abruptly : there are bulls appearing and your volume is false...
-Ensure that the full volume of sample was properly drawn into the tip.
-Withdraw the tip from the sample.
-To dispense the liquid, gently touch the tip to the side of the receiving vessel, immersing the tip into liquid within the vessel. Press the plunger to the SECOND stop.
-With the plunger fully pressed, withdraw the tip carefully, wiping residual drops against the vessel wall.
-Allow plunger to return to the UP position.
-Discard the tip by depressing the tip ejector button.

Note down that different tips exist : ensure that you have the right one (labels will indicate you the size, etc.). It's better to use filter tips.
To train, you can simply pipette water : it's important to know how much 1 µl is...

To be continued...

Growing bacteria in liquid medium

-Light the Bunsen burner. It permits you to keep a 10 cm perimeter sterile et thus not to contaminate your future colonies.
-Get a 50mL Falcon tube and put into 5 mL of LB medium. Add supplementary stuff if needed (antibiotics, metabolites, etc.).
-Pick up a sterile toothpick. Use it to gather a single colony of cells (you know, a white point on your Petri dish...).
-Place the toothpick with the colony into the solution.
-Incubate overnight at 37°C with shaking (at about 200 rpm).

Next morning, after a cup of coffee and a croissant, you can check up .
<<home

Strains

Here you can find the list of strains we have.

E. Coli MG1655

WT

E. Coli w121

We got the w121 strain from a lab in Pasteur Institute. This strain is [DapA-; Erythromycin R], but also has a couple of other mutations we are not interested in.

E. Coli Ftsz -TS84

Three clones are available : 121.1, 121.2, 121.3. More details soon

Acinetobacter: dehydrated strain

Transduction with P1 bacteriophage

Preparation of the P1 stock on the w121 strain.

Filtration of the CaCl₂ and MgSO₄ stocks
Launch a culture:

- 15ml LB

- 225µl Cacl2

- 450µl MgSO4

- 90µl DAP

- 150µl W121 from an ON culture

Let grow until OD=0.2 (~1h)
Add 100µl P1 (stock 10¹⁰/ml)
Incubate 2h
Add 200µl Chloroform
Vortex, centrifuge (15min @ 3000rpm)
Recover the supernatant + add 200µl Chloroform = Stock keep at 4°C

Transduction to MG1655 using the P1 stock made on w121.

First step for transduction : adsorption

- Control (1mL LB MgSO4 30mM; CaCl2 15mM)
- 5µL Phage + 900µL LB (MgSO4 30mM; CaCl2 15mM) + 100µL MG1655 Culture overnight
- 50µL Phage + 900µL LB (MgSO4 30mM; CaCl2 15mM) + 100µL MG1655 Culture overnight
- 500µL Phage + 500µL LB (MgSO4 30mM; CaCl2 15mM) + 100µL MG1655 Culture overnight

20 min at 37°C
Centrifuge at 12000rpm during 5 min
Resuspend bacteria in 1,5mL LB (NaCitrate 20mM; DAP 300µM)
Incubation 1h10 at 37°C with agitation
Centrifuge 5 min at 5000rpm
Plate bacteria on LB-agar (NaCitrate 20mM; DAP 300µM)

Titration of bacteriophages

Take your stock of bacteriophage
make several dilution of your stock, for example :
- 10µL of stock in 990µL MgSO₄ 0.1M -> d2
- 10µL of former solution in 990µL MgSO₄ 0.1M ->d4
- 10µL of former solution in 990µL MgSO₄ 0.1M ->d6
- 10µL of former solution in 990µL MgSO₄ 0.1M ->d8
In a petri dish containing LB, spread a solution of 100µL of E. Coli MG1655 in stationnary phase + warm top agar 900µL (TA,7)
Spread this mix over a petri dish
add droplets (7µL) of your phages dilution on the petri dish, mark the places where you put these droplets
Incubate ON at 37°C
check for lyse plaque

Preparation of DAP solution from the powder (50mM)

Step of Friday, July 6

M(DAP)=190.2g/mol
I put 0.285g of DAP in 30ml water
Aliquoted by 15ml
Stored in the freezer at -20°C
the stock is 166x

Preparing growth media

Making 10 petri dish (LB+tet+citrate+DAP)

take 250ml of LB
warm it up in the microwave for ~ 6min
wait until you can handle the bottle for 2sec
add 5ml of citrate 1M
add 1.5ml of DAP
add 250µL of tetracycline (stored in freezer at 1000x)
spread the medium in about 10 petri dish

Making 10 petri dish (LB+erythromycin+citrate+DAP)

take 250ml of LB
warm it up in the microwave for ~ 6min
wait until you can handle the bottle for 2sec
add 5ml of citrate 1M
add 1.5ml of DAP
add 1.9mL of erythromycin (stored in the freezer at 133x)
spread the medium in about 10 petri dish

Low Nitrogen Minimum Medium

The LNMM medium contains:

2g KH₂PO₄
1.18g succinic acid
0.1g NH₄SO₄
980mL H₂O
water adjudted to pH 7.0 with KOH

After autoclaving, 20mL of sterile MgSO₄ 2% (w/v) are added.
Solid media were prepared by the addition of 1.5% (w/v) agar-agar.
Culture were incubated at 30°C aerobic.

Nile Red

Sigma (N3013)

The dye is used at 0.5µg / mL medium. (liquid or solid).

make a solution of 0.25mg Nile red / mL DMSO
take 0.002 mL/mL medium from the previous solution (final concentration 0.5µg Nile Red/ mL medium).

This dye is excited by light around 450-500 nm and the emission light is > 528 nm (red). It can be exited around 515-560 and emission light is >590.

Solid M9 Minimum Medium

Prepare M9 (5x):

- 64 g Na2HPO4.7H2O - 15 g KH2PO4 - 2.5 g NaCl - 5 g NH4Cl - dissolved in deionized water to a final volume of 1 L

Autoclaving

Component adding (final concentration):

- 2mM MgSO4
- 0.1mM CaCl2
- 0.4% carbon source (glycerol, glucose, lactose)
- B1 vitamine (1mM final)
- agarose (15% final)
- in sterile H2O

Chemical transformation

This protocol is based on the Invitrogen protocol (Subcloning Efficiency Competent cells). It uses DH5alpha E.coli strain chimically competent.

Put 50µL bacteria into 1.5mL microcentrifuge tube and incubate within ice.
Add 1-5µL (1-10ng) of DNA to the cells and mix gently (do not mix by pipetting up and down!)
Incubate on ice for 30 minutes
Heat shock cells for 20 seconds in a 42°C water bath without shaking
Place tubes on ice for 2 minutes
Add 950µL of pre-warmed medium of choice to each tube
Incubate tubes at 37°C for one hour at 225rpm
Spread 20 to 200µL from each transformation on pre-warmed selective plate.
Store the remaining transformation reaction at 4°C
Incubate plates overnight at 37°C

Glycerol Stock

add 1mL of 40% glycerol into cryogenic tube
add 1mL of overnight bacterial culture
vortex gently
stock at -80°C

Recombineering/Lambda red-mediated gene replacement

See OpenWetWare http://openwetware.org/wiki/Recombineering/Lambda_red-mediated_gene_replacement

This method is based on homologous recombination using lambda red system. The knocked out gene is replaced with antibiotic resistance gene (kanamycin or chloramphenicol) that is generated by PCR by using primers with 50 nt hommolgy extension. This is accomplished by Red-mediated recombination in this flanking homologies. After selection, the resistance gene can be eliminated by using a helper plasmid expressing the FLP recombinase which acts on the directly repeated FRT sites flanking the resistance gene. The red and FLP helper plasmid can be simply cures by growth at 37°C because they are temperature sensitive replicons.

E.coli is not transformable with linear DNA because of the presence of intracellular exonucleases that degrade linear DNA (exonuclease V of the RecBCD recombination complex). The viral Lambda Red system includes 3 genes: gamma, beta and exo. Gam inhibits the RecBCD exonuclease V so that Bet and Exo can gain access to DNA ends to promote recombination.

Miniprep

We used the Wizard Plus SV Minipreps DNA Purification System (Promega) protocol

Production of cleared lysate

pellet 1-10mL overnight culture for 5 minutes
thoroughly resuspend pellet with 250µL Cell Resuspension Solution
add 250µL Cell lysis solution to each sample: invert 4 times to mix
add 10µL Alkaline protease solution to each sample: invert 4 times to mix. Incubate 5 minutes at room temperature
add 350µL Neutralization Solution; invert 4 times to mix
centrifuge at top speed for 10 minutes at room temperature

Binding of plasmid DNA

insert Spin Column into collection tube
decant cleared lysate into spin column
centrifuge at top speed for one minute at room temperature. Discard flowthrough and reinsert column into collection tube.

Washing

add 750µL Wash solution. Centrifuge at top speed for 1 minute. Discard flowthrough and reinsert column into collection tube
repeat with 250µL wash solution
centrifuge at top speed for 2 minutes at room temperature

Elution

transfer spin column to a sterile 1.5mL microcentrifuge tube, beeing careful not to transfer any of the column wash solution with the spin column. If the spin column has column wash solution associated with it, centrifuge again for 1 minute at top speed, then transfer the speed column to a new, sterile microcentrifuge tube
add 100µL of nuclease free water to the spin column. Centrifuge at top speed for 1 minute at room temperature.
discard column and store DNA at -20°C or below.

Fluorescent single cells visualisation

Slide preparation

Make LB-agarose : 0.15g of agarose in 10ml LB
warm it up in the microwave (a lot!!, to avoid cristal of agarose that could remain in the gel => ugly over microscope)
wash the slide (special slide with "two holes" with ethanol)
put ~80µL of LB-agarose in each hole
spread the gel with another slide and wait for ~2 min
remove extra gel with a cutter
put a droplet on the gel:

if you have a solid culture:

- pick up some colonies

- suspend within 100µL liquid medium (M9)

- put 1-2 µL on the gel

if you have an avernight liquid culture:

- take 1mL of the culture

- centrifugate 1000 rpm 1 min

- resuspend into 150 µL of medium (M9)

- put 1-2 µL on the gel slide

spread it by moving the slide
wait until you don't see the droplet anymore (2min)
put a coverslip and attach it with nailpolish
optionally : you can put wax around

Turning on the microscope

turn on : the light, the microscope, the shutter, the joystick
launch Metamorph
turn up the condenser and the objectives in load position
!! take care : not keeping the light on if we doesn't use it
!! do not switch on short after
wait 5 min (the microscope is checking the ranges of movement

Visualisation

Preparation

put a droplet of immersion oil on your slide, and attach it on the slide
Journal >> Show task bar

Task bar

>> Trans ON/OFF: turn on the halogen lamp

>> Binocular/Camera: turn on the binocular or the camera

>> Choose the fiter

Move up the objective until oil touches the slides
Turn on the binocular with trans filter
Move up the stage with micrometric "screw" until you see your cells, or something moving
Acquire:

- Digitizer (10MHz!!)

- Image >> New !!! ( without this, each image you take will overwrite)

- Settings >> (trans ou fluo; choice of exposure time)

Turn on the camera (Task bar >> Camera)

Trans Image

Turn trans light on (Task bar >> Trans ON) and trans filter

Acquire >> 100ms trans

Acquire >> Show Live

Acquire >> Acquire: acquire the trans image

Fluo image

Task bar >> fluo filter; trans OFFOFF)

Acquire >> 1s fluo

Acquire >> Acquire: acquire the fluo image

Acquire your images, etc, have fun
save your images in E:\iGEM\YYYY-MM-DD\a_proper_name

Turning off the microscope

Turn off the software, the microscope and the differents machines

NB : Nile Red, choose mRFP1/Texas Red filter

<<home

Digestion reactions

In order to perform digestion reactions, we use enzymes purchased from "New England Biolabs".

A classical digestion reaction is performed as follows:

Prepare a reation mix (50µL reaction volume):

1µg of template DNA
0.5µL BSA 100x (some enzymes require Bovine Serum Albumin in order to be functional)
5µL reaction buffer 10x (see below)
2-4µL of enzyme(s) (no more than 10% of total volume)
H2O qsp 50µL

Enzymes function in different buffers. In order to perform double digestion reactions, buffer must be chosen with care, (EnzymX program provides information on buffer compatibility). The informadtion provided on NEB website is usefull:

More details on double digest buffers :

http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/double_digests.asp

More details on double digest buffers including EcoR1:

http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/buffer_activity_unique_re.asp

- 2.5 hours at 37°C to digest
- 20 mins at 80°C to heat inactivate enzyme
- 4°C forever

Preparation de gel d'agarose

agarose "entre 1 et 3%" 2 et 3% pour très petit fragment (petit = 200paires de bases) en routine 1.25 :

3g d'agarose+ 200 mL de TBE 0.5 x(trisborate EDTA)

-faire chauffer au micro-onde en surveillant parce que ca rentre en ebullition : arreter attendre et relancer -quand la poudre est fondue, sortir du micro onde, et rajouter 14µL de BET (manipuler avec precaution car toxique)

-couler encore chaud dans les moules: petits gels, et grand gels, petit puits(gels analytique) et grand puit pour des gels de purification( jusqu a 30 µL)

a conserver dans du TBE 0.5 x

@@ Line 60: / Line 60: @@
 ==Transduction with P1 bacteriophage ==
 === Preparation of the P1 stock on the w121 strain. ===
-Step of  Tuesday, July 3
 * Filtration of the CaCl<sub>2</sub> and MgSO<sub>4</sub> stocks
 * Launch a culture:
-** 15ml LB
+:- 15ml LB <br>
-** 225µl Cacl2
+:- 225µl Cacl2<br>
-** 450µl MgSO4
+:- 450µl MgSO4<br>
-** 90µl DAP
+:- 90µl DAP<br>
-** 150µl W121 from an ON culture
+:- 150µl W121 from an ON culture<br>
 * Let grow until OD=0.2 (~1h)
 * Add 100µl P1 (stock 10<sup>10</sup>/ml)
@@ Line 77: / Line 76: @@
 === Transduction to MG1655 using the P1 stock made on w121. ===
-Step of Wednesday, July 4
-''To be completed...''
+* First step for transduction : adsorption<br>
+- Control (1mL LB MgSO4 30mM; CaCl2 15mM)<br>
+- 5µL Phage + 900µL LB (MgSO4 30mM; CaCl2 15mM) + 100µL MG1655 Culture overnight<br>
+- 50µL Phage + 900µL LB (MgSO4 30mM; CaCl2 15mM) + 100µL MG1655 Culture overnight<br>
+- 500µL Phage + 500µL LB (MgSO4 30mM; CaCl2 15mM) + 100µL MG1655 Culture overnight<br>
+*20 min at 37°C<br>
+*Centrifuge at 12000rpm during 5 min<br>
+*Resuspend bacteria in 1,5mL LB (NaCitrate 20mM; DAP 300µM)<br>
+*Incubation 1h10 at 37°C with agitation<br>
+*Centrifuge 5 min at 5000rpm<br>
+*Plate bacteria on LB-agar (NaCitrate 20mM; DAP 300µM)<br>
 === Titration of bacteriophages ===
@@ Line 193: / Line 201: @@
 * vortex gently
 * stock at -80°C
+== Recombineering/Lambda red-mediated gene replacement ==
+See OpenWetWare [[http://openwetware.org/wiki/Recombineering/Lambda_red-mediated_gene_replacement]]
+This method is based on homologous recombination using lambda red system. The knocked out gene is replaced with antibiotic resistance gene (kanamycin or chloramphenicol) that is generated by PCR by using primers with 50 nt hommolgy extension. This is accomplished by Red-mediated recombination in this flanking homologies. After selection, the resistance gene can be eliminated by using a helper plasmid expressing the FLP recombinase  which acts on the directly repeated FRT sites flanking the resistance gene. The red and FLP helper plasmid can be simply cures by growth at 37°C because they are temperature sensitive replicons.
+[[Image: Wanner_scheme.jpg|center|400px]]
+E.coli is not transformable with linear DNA because of the presence of intracellular exonucleases that degrade linear DNA (exonuclease V of the RecBCD recombination complex). The viral Lambda Red system includes 3 genes: gamma, beta and exo. Gam inhibits the RecBCD exonuclease V so that Bet and Exo can gain access to DNA ends to promote recombination.
 == Miniprep ==
@@ Line 332: / Line 350: @@
 <br>
 - 4°C forever
-==preparation de gel d'agarose==
+==Preparation de gel d'agarose==
 agarose "entre 1 et 3%"

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