Team:TUDelft/Protocols

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{{Template:TUDelftiGEM2009}}
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__TOC__
__TOC__
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=Protocols=
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='''Protocols'''=
==Making cells competent==
==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.
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.
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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==
 +
[[#Protocols | Back to top]]<br>
'''Materials'''
'''Materials'''
*Plate of cells to be made competent
*Plate of cells to be made competent
-
*[[TMF|TMF buffer]]
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*TMF buffer
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*[[LB|LB media]]
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*LB media
*Ice
*Ice
'''Glassware & Equipment'''
'''Glassware & Equipment'''
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'''Preparation'''
'''Preparation'''
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#Grow a 5ml [[Bacterial cell culture|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 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 OD<sub>600</sub> of 0.5 - 0.8.  
#Grow the diluted culture to an OD<sub>600</sub> 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&deg;C but if you have just made it fresh then put it in an ice bath).
+
#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&deg;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.
#Split the culture into two 50ml falcon tubes and incubate on ice for 10 min.
'''All subsequent steps should be carried out at 4&deg;C and the cells should be kept on ice wherever possible'''
'''All subsequent steps should be carried out at 4&deg;C and the cells should be kept on ice wherever possible'''
#Centrifuge for 15 minutes at 4000 rpm and 4&deg;C.
#Centrifuge for 15 minutes at 4000 rpm and 4&deg;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.
#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.
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#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 &mu;l aliquots to your chilled eppendorfs.
#Add 100 &mu;l aliquots to your chilled eppendorfs.
#Flash freeze the eppendorfs containing the cells with liquid nitrogen.
#Flash freeze the eppendorfs containing the cells with liquid nitrogen.
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#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.
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==Restrictions==
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==Preparing electro-competent cells==
[[#Protocols | Back to top]]<br>
[[#Protocols | Back to top]]<br>
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Try to do a restriction in a relatively large volume. As a rule of thumb, use a volume of 50 ul / 500 ng DNA.
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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
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* Calculate the amount of DNA you want to use
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==Electroporation==
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* add H2O
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[[#Protocols | Back to top]]<br>
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* add 10 x H buffer (Roche)
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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
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* add your calculated amount of DNA
+
 
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* add 0.5 ul of each enzyme. Keep in mind 0.5 ul = 5 U, where 1 U is defined as the amount of enzyme cutting 1000 ng of DNA / hour, so for extremely large amounts of DNA adjust this.
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==Restrictions and Ligations==
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* keep on 37°C for 2-3 hours.
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[[#Protocols | Back to top]]<br>
 +
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==
==Purifying small DNA parts==
[[#Protocols | Back to top]]<br>
[[#Protocols | Back to top]]<br>
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''Protocol found on OpenWetWare''
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We used standard Qiagen PCR purification kit.
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This protocol is for a simple ethanol precipitation of small fragments.  This protocol was used to (partially) purify a DNA fragment containing a ribosome binding site (~40 bp) during 3A assembly].  The fragment was generated via restriction digest and it was used in a ligation reaction.  Note that this protocol simply concentrates your sample and removes enough salts/enzymes for ligation to be successful.  All DNA fragments from your digest will still be present in your pellet.  These residual DNA fragments do not matter for 3A assembly which selects against incorrect ligation products.
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-
 
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===Materials===
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-
 
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*Absolute Ethanol (100% = 200 proof)
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*95% ethanol
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*Tabletop centrifuge
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*-80&deg;C freezer
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-
 
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===Procedure===
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#Add 2 volumes ice cold absolute ethanol to sample.  <br> Generally the sample is in a 1.5 mL eppendorf tube.  I recommend storing the absolute ethanol at -20&deg;C.
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#Incubate 1 hr at -80&deg;C.  <br> The long incubation time is critical for small fragments.
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#Centrifuge for 30 minutes at 0&deg;C at maximum speed (generally >10000 g at least).
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#Remove supernatant.
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#Wash with 750-1000 &mu;L room-temperature 95% ethanol.  <br> Another critical step for small fragments under 200 base pairs.  Generally washing involves adding the ethanol and inverting several times.
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#Centrifuge for 10 minutes at 4&deg;C at maximum speed (generally >10000 g at least).
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#Let air dry on benchtop. <br> I generally let the pellet air dry completely such that it becomes white so that all residual ethanol is eliminated.
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#Resuspend in an appropriate volume of H<sub>2</sub>O. <br> Many protocols recommend resuspending in 10 mM Tris-HCl or TE.  The advantage of TE is that EDTA chelates magnesium ions which makes it more difficult for residual DNases to degrade the DNA.  I generally prefer H<sub>2</sub>O and don't seem to experience problems of this sort.  If you plan to ultimately use electroporation to transform your DNA then resuspending in H<sub>2</sub>O has the advantage of keeping the salt content of your ligation reaction down.
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==DNA precipitation==
==DNA precipitation==
[[#Protocols | Back to top]]<br>
[[#Protocols | Back to top]]<br>
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Another protocol for DNA precipitation, it was used to concentrate DNA samples for sequencing.
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Standard Qiagen Minprep Kit was used for plasmid isolation.
-
 
+
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* Add 1/10 volume of 3M Sodium Acetate (NaAc), pH 4.8
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* Add 2 volumes of 96% ethanol (EtOH)
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* Store for at least 1h @ -20ºC or 20' @ -80ºC (can also be stored o/n)
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* Spin for 20' at max speed and 4ºC
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* Decant supernatant and wash pellet with 1.5 volume of 70% EtOH (EtOH has to be cold)
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* Spin for 10' at max speed and 4ºC
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* Decant supernatant and air-dry pellet in approximately 15' (no EtOH should be left)
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* Resuspend pellet in wanted volume of H<sub>2</sub>O or TE
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* Incubate for 10' @ 4ºC to ensure all DNA is dissolved
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* NanoDrop for concentration and store at -20ºC for later use
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-
 
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==Ligation==
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-
[[#Protocols | Back to top]]<br>
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First make sure you have purified the DNA after restriction. Ligation should be in a small volume (we usually use 15 ul), so elute your DNA from the column in a small volume/high concentration.
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* add H2O
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* add 10 x ligation buffer
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* add backbone and insert (theoretically in a 1:3 or 1:4 ratio, for 3A assembly it seemed to work at 1:1 ratios,  possibly even better). DNA amounts added are at least 50 ng of the backbone and if possible 100-150 ng of the insert DNA (including it's backbone).
+
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* add 1 ul of T4 Ligase.
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* keep the reaction at 16ºC for at least 2 hours, but o/n is preferable.
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* if used for transformation, all DNA can be added to competent cells, or if you want to analyze it on gel, keep 5 ul.
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-
==PCR==
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==Colony PCR==
[[#Protocols | Back to top]]<br>
[[#Protocols | Back to top]]<br>
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===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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===Gradient PCR===
 
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Gradient PCR is mainly used to determine the best annealing temperature for primers. This is done in this project with Taq polymerase mastermix, as this is cheaper than ''Pfx''. However, as long as a PCR machine capable of making gradients is present, a gradient PCR can be performed with any polymerase. During the annealing step (step 3 in the taq mastermix protocol) every column in the PCR machine has a different temperature, going up from left to right. The range of the gradient can be installed manually, however the actual temperatures cannot (at least not in our machine). An example of PCR products put on gel after a gradient PCR can be seen in the lab notebook at the 20th of August, where gradients of 5ºC in 12 steps were tested for the atoB, idi and ispA primer pairs.
 
-
 
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===Touchdown PCR===
 
-
 
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Some of the ordered primers had long sequences that are not supposed to bind to the target DNA (the pre- and suffix for forward and reverse primer, respectively). Here low annealing temperatures could lead to a lot of aspecific product formation, while high annealing temperatures could be too specific, causing very little product formation. To suppress this, a touchdown PCR can be performed. Again 50 - 100 ng of template DNA should be used and any polymerase. The PCR program used in this project, with ''Pfx'' polymerase, looked like this:<br>
 
-
1. 5' @ 94°C<br>
 
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2. 1' @ 94°C<br>
 
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3. 1' @ 65°C --> temperature is lowered with 0.5°C per cycle<br>
 
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4. 3' @ 72°C<br>
 
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5. go to 2, 20 cycles in total<br>
 
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6. 1' @ 94°C<br>
 
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7. 1' @ 94°C<br>
 
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8. 3' @ 72°C<br>
 
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9. go to 6, 20 cycles in total<br>
 
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10. 7' @ 72°C<br>
 
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11. ∞ @ 10°C<br><br>
 
==DNA gels==
==DNA gels==
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* Let the gel run at a voltage between 60V and 120V, depending on desired resolution/time available.
* 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.
* 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.
-
 
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==Protein content measurement==
 
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[[#Protocols | Back to top]]<br>
 
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===BC assay===
 
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The BCA kit used is of the company Uptima.
 
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* Make a dilution series of standard 2mg/ml bovine serum albumin (BSA). We used 2, 1, 0.75, 0.5, 0.25, 0.1, 0.02 and 0 mg/ml.
 
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* Pipet 25ul of every sample from the standard solutions to a well in a 96-wells plate to make a calibration curve. Also pipet 25ul of every sample with unknown protein content. Always load samples at least twice.
 
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* Add 1 ml of reagens B to 50 ml of reagens A and mix.
 
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* Add 200ul of AB mix to all wells that have a sample in them.
 
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* Incubate 30' @ 37ºC
 
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* Read out OD<sub>562</sub> in plate reader
 
==Fluorescence Measurements==
==Fluorescence Measurements==
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==Protein Precipitation==
 
[[#Protocols | Back to top]]<br>
[[#Protocols | Back to top]]<br>
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During the project, several ways of protein precipitation were used. Here is an overview of all of them.
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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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*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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===Perchloric Acid (PCA)===
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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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* Add 1 volume of 1M PCA to sample and mix
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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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* Spin for 20' @ 1,500g and 4ºC
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*The program does the following:
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* Remove supernatant and spin again for 20' @ 1,500g and 4ºC
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**Set Temperature to 37&deg;C
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* Remove the supernatant as much as possible and resuspend in wanted volume of H<sub>2</sub>O
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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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===Acetone/Trichloric Acid (TCA)===
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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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* Mix 10 volumes of cold 10% TCA in acetone (stored @ -20ºC) with your samples, vortex, and incubate at -20ºC for at least 3h, but o/n is optimal
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* Spin samples 10' @ 15,000g and remove supernatant
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* Wash pellet with 10 volumes of acetone, vortex, and incubate for at least 10' at -20ºC
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* Spin 5' @ 15,000g, remove supernatant (carefully) and air dry pellets
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* Resuspend in wanted volume of H<sub>2</sub>O
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===TCA/Deoxycholate (DOC)===
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* Add 1/100 volume of 2% DOC, mix, and incubate on ice for 30'
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* Add 100% TCA so that final concentration of TCA in the sample is 15%
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* Vortex immediately to avoid formation of large conglomerates that can trap contaminants
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* Keep the sample on ice for at least 1h to allow protein to precipitate, but prefarably o/n
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* Spin 10' @ 15,000g and remove supernatant as much as possible
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* Wash pellet with EtOH or Acetone (stored @ -20ºC)
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* Vortex and incubate at RT for 5'
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* Spin for 10' @ 15,000g and remove supernatant
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* Repeat the last three steps (wash pellet twice)
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* Dry pellet (we let it air dry, although the original protocol suggested to do it under a SLOW stream of nitrogen)
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* Resuspend in wanted volume of H<sub>2</sub>O
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===Methanol (MeOH)/Chloroform===
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* Add 4 volumes of MeOH and vortex well
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* Add 1 volume of chloroform and vortex
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* Add 3 volumes of dH<sub>2</sub>O and vortex
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* Spin 2' @ 15,000g - the sample will divide in two phases, proteins should be at the liquid interface
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* Remove aqueous top layer, add 4 volumes of methanol and vortex
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* Spin 2' @ 15,000g
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* Remove supernatant as much as possible
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* Air dry pellet (again, original protocol mentioned drying under nitrogen or speed-vacuum)
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* Resuspend in wanted volume of H<sub>2</sub>O
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==Cell Lysis==
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[[#Protocols | Back to top]]<br>
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===Promega lysis buffer===
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* Spin off 1ml of culture for 5' @ 10,000 rpm and 4ºC
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* Decant sample and get out as much of the LB medium as possible
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* Resuspend pellet in 1ml of 1x lysis buffer in H<sub>2</sub>O
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* Incubate for 30' on ice
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* Spin off 2' @ max speed and 4ºC
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* Transfer supernatant (with protein) to a fresh eppendorf tube
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===Bead beater===
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* Spin off 1ml of culture for 5' @ 10,000 rpm and 4ºC
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* Decant sample and get out as much of the LB medium as possible
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* Resuspend pellet in 1ml of 1x PBS
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* Add 0.5g of small acid-washed glass beads
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* Add 20ul of 2uM lysozyme
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* Put samples in the bead beater for 1h in the cold room
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* Spin off 2' @ max speed and 4ºC
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* Transfer 600ul of supernatant (with protein) to a fresh eppendorf tube
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===Fastprep===
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* Spin off 1ml of culture for 5' @ 10,000 rpm and 4ºC
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* Decant sample and get out as much of the LB medium as possible
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* Resuspend pellet in 1ml of 1x PBS
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* Add autoclaved glass bead (d=1mm) to the sample, the amount needed equals the amount filling the conical part at the bottom of a 2 ml Greiner Bio1 microcentrifuge tube
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* Shake the sample 5s at intensity 5 in the Thermo Savant FastPrep FP120 Homogenizer
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* Spin off 2' @ max speed and 4ºC
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* Transfer 500 ul supernatant (with protein) to a fresh eppendorf tube
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-
 
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===Sonication===
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* Spin off 1ml of culture for 5' @ 10,000 rpm and 4ºC
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* Decant sample and get out as much of the LB medium as possible
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* Resuspend pellet in 1ml of 1x PBS
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* Sonicate samples 2 times for 15 seconds with a 15 second pause in between. Make sure samples are kept on ice during sonication.
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* Spin off 2' @ max speed and 4ºC
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* Transfer supernatant (with protein) to a fresh eppendorf tube
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-
 
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=Bacterial transformation=
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==Introduction==
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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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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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==Protocols==
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OpenWetWare already has a number of protocols relating to bacterial transformation '''but more are always welcome'''.
+
-
 
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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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===Chemical transformation===
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If you plan on doing a chemical transformation, then you should see these pages -
+
-
 
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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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-
 
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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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-
 
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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]]
+
-
 
+
-
==References==
+
-
 
+
-
<biblio>
+
-
#MolecularCloning isbn=0-87969-577-3
+
-
# Hanahan91 pmid=1943786
+
-
# Nishimura90 pmid=2235524
+
-
# Hanahan89 US Patent 4,851,348 [[Media:pat4851348.pdf]]
+
-
# Jessee90 US Patent 4,981,797  [[Media:pat4981797.pdf]]
+
-
# Donahue01 US Patent 6,247,369 [[Media:pat6274369.pdf]]
+
-
# Greenr04 US Patent 6,706,525 [[Media:pat6706525.pdf]]
+
-
# Donahue04 US Patent 6,709,854 [[Media:Pat6709854.pdf]]
+
-
# Bloom04 US Patent 6,709,852 [[Media:pat6709852.pdf]]
+
-
# Bloom05 US Patent 6,855,494 [[Media:pat6855494.pdf]]
+
-
# Jessee05 US Patent 6,960,464 [[Media:pat6960464.pdf]]
+
-
# Cohen-PNAS-1972 pmid=4559594
+
-
# Sharma07 Sharma AD, Singh J, Gill PK; Ethanol mediated enhancement of bacterial transformation, Electronic Journal of Biotechnology (2007), 10(1), DOI: 10.2225/vol10-issue1-fulltext-10, [http://www.ejbiotechnology.info/content/vol10/issue1/full/10/index.html here].
+
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+
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</biblio>
+
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[[Category:Protocol]]
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[[Category:In vivo]]
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[[Category:Escherichia coli]]
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=Medium preparation=
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='''Media, Buffers and Stcoks preparation'''=
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==Buffers & (Stock) Solutions==
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[[#Protocols | Back to top]]<br>
[[#Protocols | Back to top]]<br>
===Antibiotics (1000x stock solutions)===
===Antibiotics (1000x stock solutions)===
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*10 g NaCl
*10 g NaCl
*adjust pH to 7.0
*adjust pH to 7.0
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===TSS buffer===
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For 50 mL:
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* 5g PEG 8000
 +
* 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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 +
===TMF buffer===
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For 50 mL:
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* 100mM CaCl2
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* 50mM RbCl
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* 40mM MnCl2
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* Add ddH2O to 50 mL
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Filter sterilize (0.22 μm filter) TMF buffer and store at 4ºC or -20ºC
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 +
===Basal Minimal Medium===
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For 1 litre:
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*K2HPO4 - 9 g
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*KH2PO4 - 3 g
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*(NH4)SO4 - 2 g
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*NaCitrate - 0.5 g
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*MgSO4 10x (10gr/L) (1%)
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*Glucose 10x 20%
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*vitamin B1 (thiamine) 200x 2 mg/mL
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*Amino Acids 20x 10 mg/mL
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 +
Add all except glucose solution and mix well in ddH2O.
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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===
 
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Adapted From:
 
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J. Sambrook, D.W. Russell, ''Molecular Cloning: A Laboratory Manual'' (Cold Spring Harbor Laboratory Press, New York, ed. 3, 2001) pg. A2.2
 
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[[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

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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

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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

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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

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

Electroporation

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For doing the electroporation on the electro-competent cells we used this 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

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We used standard Qiagen PCR purification kit.

DNA precipitation

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Standard Qiagen Minprep Kit was used for plasmid isolation.

Colony PCR

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  • 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

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  • 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

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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°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

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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.