Team:TUDelft/Protocols

From 2009.igem.org

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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.
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, for 50 mL:
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You will need TSS Buffer.
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** 5g PEG 8000
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** 1.5 mL 1M MgCl2 (or 0.30g MgCl2*6H20)
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** 2.5 mL DMSO
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** Add LB to 50 mL
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Filter sterilize (0.22 μm filter) TSS buffer and store at 4ºC or -20ºC
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Preparing the cells:
Preparing the cells:
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==Prepering chemically competant cells - TMF Buffer==
==Prepering chemically competant cells - TMF Buffer==
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[[#Protocols | Back to top]]<br>
'''Materials'''
'''Materials'''
*Plate of cells to be made competent
*Plate of cells to be made competent
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#Store the cells at -80&deg;C.
#Store the cells at -80&deg;C.
#It is a good idea to run a positive control on the cells.
#It is a good idea to run a positive control on the cells.
 +
 +
==Preparing electro-competent cells==
 +
[[#Protocols | Back to top]]<br>
 +
For making the electro-competent cells we used this [http://openwetware.org/wiki/Knight:Preparing_electrocompetent_cells protocol] from openwetware.org
 +
 +
==Electroporation==
 +
[[#Protocols | Back to top]]<br>
 +
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==
==Restrictions and Ligations==
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Standard Qiagen Minprep Kit was used for plasmid isolation.
Standard Qiagen Minprep Kit was used for plasmid isolation.
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==PCR==
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==Colony PCR==
[[#Protocols | Back to top]]<br>
[[#Protocols | Back to top]]<br>
-
===Colony PCR===
 
*Make biobrick mastermix, containing per sample:  
*Make biobrick mastermix, containing per sample:  
**12.5 ul ''Taq'' mastermix
**12.5 ul ''Taq'' mastermix
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*Run the iGEM colpcr program <i>(to be added later)</i>
*Run the iGEM colpcr program <i>(to be added later)</i>
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===PCR using ''Taq'' Mastermix===
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==PCR using ''Taq'' Mastermix==
 +
[[#Protocols | Back to top]]<br>
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 H<sub>2</sub>O is added to the mix.
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 H<sub>2</sub>O is added to the mix.
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7. ∞ @ 4ºC (PCR can be stopped and stored in the fridge at any time from this point on)<br>
7. ∞ @ 4ºC (PCR can be stopped and stored in the fridge at any time from this point on)<br>
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===PCR using ''Pfx'' polymerase===
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==PCR using ''Pfx'' polymerase==
 +
[[#Protocols | Back to top]]<br>
Mastermix does not exist for the ''Pfx'' polymerase. This means the components have to be added seperately. The mix consists of:
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)
* x ul template DNA (again 50 - 100 ng total)
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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''.
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''.
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==DNA gels==
==DNA gels==
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==Fluorescence Measurements==
==Fluorescence Measurements==
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[[#Protocols | Back to top]]<br>
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=Bacterial transformation=
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*The samples to be tested are cultured from plates in 2ml of the Basal Minimal Medium with appropriate antibiotics and incubated overnight at 37&deg;C at 175 rpm.  
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==Introduction==
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*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.
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Transformation is the process of introducing foreign DNA (e.g plasmids, BAC) into a bacterium.  Bacterial cells into which foreign DNA can be transformed are called '''competent'''.  Some bacteria are naturally competent (e.g ''B. subtilis''), whereas others such as ''E. coli'' are not naturally competent.  Non-competent cells can be made competent and then transformed via one of two main approaches; '''chemical transformation''' and '''electroporation'''.
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*The plate is then first read at OD600 and is then incubated again at 37&deg;C with medium shaking for around 3 hours.
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+
*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.
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There are advantages and disadvantages to both transformation methods. In general, chemical transformation is less prone to error and faster however electroporation produces a higher transformation efficiency (fraction of transformed cells that actually uptake the foreign DNA).  See [[Molecular Cloning]] for a fuller discussion of both approaches.
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*The plate reader is then read by the automatically repeating protocol with shaking at medium speed created by BioTek Synergy.
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*The program does the following:
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==Protocols==
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**Set Temperature to 37&deg;C
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OpenWetWare already has a number of protocols relating to bacterial transformation '''but more are always welcome'''.
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**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).
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** Then a delay of 100 seconds is made.
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If you use a variant on one of these protocols please feel free to add a link to your protocol from one of these pages so other users can find a protocol that works for them. Additionally, if anyone uses the Innoe or Hanahan high-efficiency protocols, then please add protocols here.
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* 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.
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+
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===Chemical transformation===
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If you plan on doing a chemical transformation, then you should see these pages -
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-
 
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*[[Preparing chemically competent cells]]
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*[[TSS|Preparing TSS buffer]]
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*[[Transforming chemically competent cells]]
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*[[Preparing chemically competent cells (Inoue)]]
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*[[Transforming chemically competent cells (Inoue)]]
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*[[TOP10 chemically competent cells]]
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[[Chemical transformation buffer comparison]]
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Someone should check out the claims of Nishimura90.  [[User:Tk|tk]] 08:58, 25 September 2007 (EDT)
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Rubidium chloride transformation protocol [http://wheat.pw.usda.gov/~lazo/methods/goldberg/competen.html here]
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Someone should check the claims of 1e10 chemical competence using 10% ethanol and calcium chloride protocols [http://www.ejbiotechnology.info/content/vol10/issue1/full/10/index.html here].
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===Electroporation===
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If you plan on using electroporation, then see these pages -
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*[[Electrocompetent cells]]
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*[[Electroporation]]
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==References==
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[[Category:Protocol]]
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[[Category:In vivo]]
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[[Category:Escherichia coli]]
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='''Media, Buffers and Stcoks preparation'''=
='''Media, Buffers and Stcoks preparation'''=
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*10 g NaCl
*10 g NaCl
*adjust pH to 7.0
*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)===
===10x TBE (Tris, Boric Acid, EDTA)===
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Adjust volume to 1L<br>
Adjust volume to 1L<br>
The pH of 1x PBS should be 7.4
The pH of 1x PBS should be 7.4
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-
 
-
===Source===
 
-
Adapted From:
 
-
 
-
J. Sambrook, D.W. Russell, ''Molecular Cloning: A Laboratory Manual'' (Cold Spring Harbor Laboratory Press, New York, ed. 3, 2001) pg. A2.2
 
-
 
-
[[Category:Material]]  [[Category:Standard Media]]
 
=Lock/key synthesis=
=Lock/key synthesis=

Latest revision as of 19:32, 21 October 2009

Contents

Protocols

Making cells competent

Back to top
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

Back to top
Standard transformation procedure

  • 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

Back to top
Materials

  • 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

  1. 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.
  2. Grow the diluted culture to an OD600 of 0.5 - 0.8.
  3. 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).
  4. 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

  1. Centrifuge for 15 minutes at 4000 rpm and 4°C.
  2. 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.
  3. 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.
  4. Add 100 μl aliquots to your chilled eppendorfs.
  5. Flash freeze the eppendorfs containing the cells with liquid nitrogen.
  6. Store the cells at -80°C.
  7. It is a good idea to run a positive control on the cells.

Preparing electro-competent cells

Back to top
For making the electro-competent cells we used this protocol from openwetware.org

Electroporation

Back to top
For doing the electroporation on the electro-competent cells we used this protocol from openwetware.org

Restrictions and Ligations

Back to top
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

Back to top
We used standard Qiagen PCR purification kit.

DNA precipitation

Back to top
Standard Qiagen Minprep Kit was used for plasmid isolation.

Colony PCR

Back to top

  • 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

Back to top
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

Back to top
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

Back to top

  • 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

Back to top

  • 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

Back to top

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[1], 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.