Team:TUDelft/Protocols

From 2009.igem.org

1 Protocols
2 Media, Buffers and Stcoks preparation
3 Lock/key synthesis
4 Conjugation Protocol

Protocols

Making cells competent

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Most of the time, we used Top10 chemically competent cells. We did make a stock of chemically competent DB3.1 cells with the following protocol (found on OpenWetWare). We found that these cells were indeed very competent.

You will need TSS Buffer.

Preparing the cells:

Grow a 5ml overnight culture of cells in LB media.
In the morning, dilute this culture back into 25-50ml of fresh LB media in a 200ml conical flask. You should aim to dilute the overnight culture by at least 1/100.
Grow the diluted culture to an OD600 of 0.2 - 0.5. (You will get a very small pellet if you grow 25ml to OD600 0.2)
Put eppendorf tubes on ice now so that they are cold when cells are aliquoted into them later. If your culture is X ml, you will need X tubes. At this point you should also make sure that your TSS is being chilled (it should be stored at 4oC but if you have just made it fresh then put it in an ice bath).
Split the culture into two 50ml falcon tubes and incubate on ice for 10 min.

All subsequent steps should be carried out at 4oC and the cells should be kept on ice wherever possible

Centrifuge for 10 minutes at 3000 rpm and 4oC.
Remove supernatant. The cell pellets should be sufficiently solid that you can just pour off the supernatant if you are careful. Pipette out any remaining media.
Resuspend in chilled TSS buffer. The volume of TSS to use is 10% of the culture volume that you spun down. You may need to vortex gently to fully resuspend the culture, keep an eye out for small cell aggregates even after the pellet is completely off the wall.
Add 100 μl aliquots to your chilled eppendorfs and store at − 80oC.

Transformations

Remove competent cells from -80, let thaw for 10 min on ice and aliquot in 50 ul amounts.
add 2-5 ul of vector, usually in H2O, to 50 ul cells, no mixing by pipet due to shear induction.
keep on ice for 20 minutes (vector spreading through volume)
heat shock (42°C) for 45 seconds
keep on ice for 2 minutes
add 200 ul SOC, put on 37°C for 1 hour or longer with agitation.
plate out 250 ul on appropriate antibiotics.

Prepering chemically competant cells - TMF Buffer

Plate of cells to be made competent
TMF buffer
LB media
Ice

Glassware & Equipment

Falcon tubes
500μl Eppendorf tubes, on ice
200ml conical flask
200μl pipetman or repeating pipettor
5ml pipette

Preparation

Grow a 5ml overnight culture of cells in LB media. In the morning, dilute this culture back into 40ml of fresh LB media with 0.8 ml of Mg-mix (0.5M Magnesium chloride + 0.5M Magnesium sulfate) in a 100ml conical flask. You should aim to dilute the overnight culture by at least 1/100.
Grow the diluted culture to an OD₆₀₀ of 0.5 - 0.8.
Put eppendorf tubes on ice now so that they are cold when cells are aliquoted into them later. If your culture is X ml, you will need X tubes. At this point you should also make sure that your TMF is being chilled (it should be stored at 4°C but if you have just made it fresh then put it in an ice bath).
Split the culture into two 50ml falcon tubes and incubate on ice for 10 min.

All subsequent steps should be carried out at 4°C and the cells should be kept on ice wherever possible

Centrifuge for 15 minutes at 4000 rpm and 4°C.
Remove supernatant. The cell pellets should be sufficiently solid that you can just pour off the supernatant if you are careful. Pipette out any remaining media.
Resuspend in 4ml chilled TMF buffer and add 1 ml of 40% glycerol. You may need to vortex gently to fully resuspend the culture, keep an eye out for small cell aggregates even after the pellet is completely off the wall.
Add 100 μl aliquots to your chilled eppendorfs.
Flash freeze the eppendorfs containing the cells with liquid nitrogen.
Store the cells at -80°C.
It is a good idea to run a positive control on the cells.

Preparing electro-competent cells

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For making the electro-competent cells we used this [http://openwetware.org/wiki/Knight:Preparing_electrocompetent_cells protocol] from openwetware.org

Electroporation

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For doing the electroporation on the electro-competent cells we used this [http://openwetware.org/wiki/Knight:Electroporation protocol] from openwetware.org

Restrictions and Ligations

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We used the New England Biolabs' Assembly Kit for this purpose and exactly followed the protocol specified by them. It works well.

Purifying small DNA parts

DNA precipitation

Colony PCR

Make biobrick mastermix, containing per sample:
- 12.5 ul Taq mastermix
- 2.5 ul 10x forward biobrick primer
- 2.5 ul 10x reverse biobrick primer
- 7.5 ul H2O
Put 25 ul in the PCR tubes.
With a toothpick or pipet point, touch a colony and stir it through the fluid
Run the iGEM colpcr program (to be added later)

PCR using Taq Mastermix

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Contents of the PCR mix is the for a large part the same as mentioned above for the Colony PCR. Differences will be noted here. First, instead of biobrick primer, any primer of choice can be added, also 2.5ul if standard solution has a concentration of 10 pmol/ul. Also x ul template DNA from a sample is added, where x depends on the total concentration of DNA in the sample. Typically 50 to 100 ng of total DNA is added. 7.5 - x ul of H₂O is added to the mix.

PCR program is:
1. 5' @ 95ºC
2. 1' @ 95ºC
3. 1' @ annealing temperature of the primer
4. 1' @ 72ºC (1' is long enough for 1kb, longer times can be used if larger products are formed)
5. repeat steps 2-4 29x (total of 30 cycles, more can be added if necessary)
6. 5' @ 72ºC
7. ∞ @ 4ºC (PCR can be stopped and stored in the fridge at any time from this point on)

PCR using Pfx polymerase

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Mastermix does not exist for the Pfx polymerase. This means the components have to be added seperately. The mix consists of:

x ul template DNA (again 50 - 100 ng total)
5.0 ul 10x buffer
2.5 ul forward primer (10 pmol/ul)
2.5 ul reverse primer (10 pmol/ul)
0.2 ul Pfx
1.5 ul dNTP's (10 mM)
1.0 ul MgSO₄ (50 mM)
37.3-x ul H₂O

The PCR program looks the same as mentioned above for Taq polymerase, only difference is the elongation temperature in step 4. This is 68ºC for Pfx.

DNA gels

Take a flask of 0.8% up to 1.5% molten agarose from the 70oC stove.
Pour a it in a taped gel tray.
Add ca. 5 ul of SYBRSafe (depending on size gel)
Add a comb and let the gel harden for ca. 15 minutes.
Remove the comb and the tape and put the gel tray in an electrophoresis tray.
Add enough 1x TBE to completely cover the gel.
Add DNA loading buffer to your samples and load them.
Let the gel run at a voltage between 60V and 120V, depending on desired resolution/time available.
Visualize the DNA by putting it in the imager for taking a picture, or if you want to cut out your DNA, put it on the blue light emitter.

Fluorescence Measurements

The samples to be tested are cultured from plates in 2ml of the Basal Minimal Medium with appropriate antibiotics and incubated overnight at 37°C at 175 rpm.
The culture is next day checked for OD600 and then diluted to 100 times in a 96 well plate by the same medium with antibiotics.
The plate is then first read at OD600 and is then incubated again at 37°C with medium shaking for around 3 hours.
The plate is then taken out and read at OD600 based on which the cultures are diluted to 10 times which must be around (0.1) with calculated samples induced with 0.1mM IPTG and 0.2mM IPTG.
The plate reader is then read by the automatically repeating protocol with shaking at medium speed created by BioTek Synergy.
The program does the following:
- Set Temperature to 37°C
- In a kinetic loop of fixed time (We used 2 hour 30 mins or 16 hour 30 mins) following measurements are taken in a time interval of 10 minutes or 20 minutes with shaking: Absorbance (600 nm filter) and Fluorescence (485nm and 520nm for GFP).
- Then a delay of 100 seconds is made.
If the protocol is programmed to generate the results in excel sheet then it is easy to get the results of the well data and interpret them.

Media, Buffers and Stcoks preparation

Antibiotics (1000x stock solutions)

Ampicillin: 100 mg/ml in H₂O
Chloroamphenicol: 34 mg/ml in etOH
Kanamycin: 10 mg/ml in H₂O
Tetracycline: 5 mg/ml etOH

SOB (Super Optimal Broth)

For 1 liter dissolve in H₂O

20 g Bacto tryptone
5 g Bacto-Yeast extract
0.5 g NaCl
10 ml 250 mM KCl
adjust pH to 7.0
before use add 5 ml of 2mM MgCl₂

SOC (Super Optimal broth with Catabolite repression)

add 20 mM glucose to 1L SOB.
You can also order small bottles from Invitrogen (which is what we did)

LB medium (Lysogeny Broth^{[http://jb.asm.org/cgi/content/full/186/3/595]}, but better known as Luria-Bertani Medium)

In 950 mL H₂O

10 g Bacto Tryptone
5 g Bacto-Yeast extract
10 g NaCl
adjust pH to 7.0

TSS buffer

For 50 mL:

5g PEG 8000
1.5 mL 1M MgCl2 (or 0.30g MgCl2*6H20)
2.5 mL DMSO
Add LB to 50 mL

Filter sterilize (0.22 μm filter) TSS buffer and store at 4ºC or -20ºC

TMF buffer

For 50 mL:

100mM CaCl2
50mM RbCl
40mM MnCl2
Add ddH2O to 50 mL

Filter sterilize (0.22 μm filter) TMF buffer and store at 4ºC or -20ºC

Basal Minimal Medium

For 1 litre:

K2HPO4 - 9 g
KH2PO4 - 3 g
(NH4)SO4 - 2 g
NaCitrate - 0.5 g

MgSO4 10x (10gr/L) (1%)
Glucose 10x 20%
vitamin B1 (thiamine) 200x 2 mg/mL
Amino Acids 20x 10 mg/mL

Add all except glucose solution and mix well in ddH2O. Autoclave and then add filter sterilized glucose solution.

10x TBE (Tris, Boric Acid, EDTA)

To make 1L, dissolve in 950 ml H₂O

54 g Tris
27.5 g Boric Acid
4.65 g EDTA or 20 ml 0.5M EDTA pH 8.0

6x DNA Gel loading buffer

Dissolve in H₂O
0.25% Bromophenolblue
0.25% Xylene Cyanol FF
40% (w/v) Sucrose

10x PBS (Phosphate Buffered Saline)

In 950 mL H₂O dissolve:

11.5g Na₂HPO₄
2g KH₂PO₄
80g NaCl
2g KCl

Adjust volume to 1L
The pH of 1x PBS should be 7.4

Lock/key synthesis

Protocol for Lock/key synthesis

Conjugation Protocol

See Conjugation Protocol.

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