Team:Valencia Biocampus/Protocols
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Protocols
- Take competent E.coli cells from –80°C freezer.
- Turn on water bath to 42°C.
- Put 100 ul of competent cells in an Eppendorf tube.
- Keep tubes on ice.
- Add 50 ng of circular DNA into E.coli cells. Incubate on ice for 10 minutes to thaw competent cells.
- Put tube(s) with DNA and E.coli into water bath at 42°C for 45 seconds.
- Put tubes back on ice for 2 minutes to reduce damage to the E.coli cells.
- Add 1 ml of LB (with no antibiotic added). Incubate tubes for 1 hour at 37°C.
- Spread about 100 ul of the resulting culture on LB plates (with Ampicillin added). Grow overnight.
- Pick colonies about 12-16 hours later.
- Prepare a 2 ml preculture in the selection medium of the strain to be transformed.
- Inoculate 20 ml of YPD for each transformation, in order to get an OD600 = 1 next day.
- inoculum vol.= (final OD)/(preculture OD)×(final vol.)/2^n
Where n is the number of divisions (generation time: 1´5 h for S. cerevisiae). - Centrifuge at 3000 rpm 5 min.
- Wash with sterile water.
- Resuspend in 30 ml of LISORB.
- Shake at ambient temperature for 30 min.
- Centrifuge at 3000 rpm 5 min and resuspend in 1 ml of LISORB. Transfer to an eppendorf tube.
- Centrifuge at 3000 rpm 5 min.
- Resuspend in 100 μl of LISORB for each transformation. Transfer 100 μL aliquots in different tubes for each transformation.
- Add 7 μl of salmon sperm DNA + 1 μl of transforming DNA.
- Incubate 10 min at ambient temperature.
- Add 260 μl of 40%PEG/LiAc/TE. Mix well.
- Incubate 1 h at 30°C.
- Add 43 μl of DMSO and give a thermal shock of 5 minutes at 42°C.
- Centrifuge at 3000 rpm 5 min.
- Wash with 1 ml of sterile water.
- Centrifuge at 3000 rpm 5 min.
- Resuspend in 0´5 ml of water and plaque:
- -50 μl
-Rest (centrifuge and decant leaving 50-100 μl). - Different cultures (each one with a different construction), which are growing in a selective media (LB + Ampicillin), get centrifuged at 4500g 5 min.
- Supernatant is removed.
- The cells can be washed (x2) with a saline solution (PBS) in order to remove impurities.
- The pellet is resuspended in 250 μL of Resuspension Solution (RNase A added to it previously. This solution is kept at 4ºC). Important: resuspend it completely.
- Transfer the suspension to an eppendorf tube.
- Add 250 μL of Lysis Solution.
- Mix it inverting the tube 4-6 times (DO NOT VORTEX!) until solution gets viscous and slightly clear.
- Important: Do not incubate more than 5 min.
- Add 350 μL of Neutralization Solution.
- Mix it inverting the tube 4-6 times. Incubate in ice for 15-30 min.
- Now if it was necessary, the process could stop here keeping the eppendorf tube in ice.
- Centrifuge 10’ (max. rpm) in order to pellet cell debris and chromosomal DNA.
- Transfer the supernatant (≈ 800 μL) to the spin column (pipetting to avoid carrying impurities).
- Important: DO NOT TRANSFERING THE PRECIPITATE!
- Centrifuge 1’.
- Flow-though liquid is removed.
- Add 500 μL of Wash Solution (Solution stock has to be perfectly closed, it contains ethanol!).
- Centrifuge ≈ 1’.
- Flow-though liquid is removed.
- 14, 15, 16 steps are repeated.
- Centrifuge 1’ in order to eliminate residual Wash Solution.
- The spin column is transferred into an eppendorf tube (the collection tube is eliminated).
- Add 50 μL of Elution Buffer to the center of spin column membrane and let it 5’ getting soaked (it increases the efficiency of process).
- Important: DO NOT CONTACT THE COLUMN MEMBRANE WITH THE PIPETTE TIP!
- Centrifuge ≈ 2’.
- To increase the efficiency (≈ 20%) we can get the flow-though liquid and repeat the steps previously described (20 and 21).
- The column is discarded and the solution which contains the purified plasmid can be stored in cold.
- Cut bands of interest from the agarose gel.
- Add 300 uL of Solution L1 for each 100 mg of gel.
- Incubate at 50ºC for 15 minutes.
- Centrifugate in a 2 ml column at 12000xg for 1 minute.
- Re-insert the spin column into the resaver tube and add 500 uL of Buffer L2.
- Centrifugate 12000xg for 1 minute.
- Discard the flow-through.
- Centrifuge 12000xg for 1 minute.
- Place the spin column into a new 1.5 mL microfuge tube.
- Add 50 uL of mQ water.
- Centrifuge 12000xg for 2 minutes.
- The digestion mixture is kept for 3 hours at 37 ° C
- The mixture is kept for 20 minutes at 80ºC
- 2 uL of EcoRI is added and incubated for 1 hour.
- 2 uL of EcoRI is added and incubated for 3 hour.
- 2 uL of PstI is added and incubated for 1 hour.
- 2 uL of PstI is added and incubated overnight
- Each colony is taken from the petri dish and resuspended in 15 μl of NaOH 20 mM in a eppendorf.
- Incubate for 15 minutes at room temperature.
- The PCR mix is prepared as shown:
- 2 ul of yeast DNA solution
5 ul of 10X PCR buffer
4 ul of dNTPs 2.5 mM
2 ul of A oligo
2 ul of B oligo
31 ul of water - Once mixed, 4 ul of 10X TAQ polymerase solution is added.
- The PCR reaction program is the next one:
- 94ºC 3 minutes
30 cycles of:- 94ºC 1 minutes
45ºC 1 minutes 30 seconds
72ºC 2 minutes
4ºC Hold - Digestion Protocol For Plasmid Backbone Using EcoRI and PstI
- Digestion Protocol For Plasmid pUC57+ Construction Using EcoRI and PstI
- Transformation Protocol Using Heat Shock
- Ligation Protocol
- Mini-preps. Purification protocol.
- Protocol for Gel Extraction
Contents |
Transformation protocols
Heat Shock Protocol for bacteria transformation
Yeast transformation
DNA extraction and purification protocols
Mini-prep
Protocol for Gel Extraction
DNA digestion and ligation protocols
Digestion Protocol For Plasmid Backbone Using EcoRI and PstI
DNA linearized plasmid Backbone (25 ng/uL) | 8 uL | |
PstI | 1 uL | |
EcoRI | 1 uL | |
Buffer 10x must be a common buffer for
EcoRI and PstI (e.g. buffer H in Roche system) |
2.5 uL | |
mQ Water | x uL | |
TOTAL | 25 uL |
Mix by pipetting when both enzymes have been added. Avoid vortexing. Enzymes are kept in cooler or ice throughout all experiments.
Digestion Protocol For Plasmid pUC57+ Construction Using EcoRI and PstI
Plasmid DNA | 96 uL | |
PstI | 2 uL + 2 uL | |
EcoRI | 2 uL + 2 uL | |
Buffer 10x must be a common buffer for
EcoRI and PstI (e.g. buffer H in Roche system) |
12 uL | |
mQ Water | 4 uL | |
TOTAL | 120 uL |
The volume used is so high because after digestion we were going to purify the different inserts from an agarose gel.
In order to optimize the digestion reaction we follow these steps
Ligation
Colony PCR
Biobricks protocols
Media protocols
LB broth for bacteria
LBA broth
LB + Chloramphenicol broth
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=== YP broth ===