From 2012.igem.org

Protocols

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Contents 1 General Protocols 1.1 Transformation protocols 1.1.1 Heat Shock Protocol for bacteria transformation 1.1.2 Yeast transformation 1.2 DNA extraction and purification protocols 1.2.1 Mini-prep 1.2.2 Protocol for Gel Extraction 1.3 DNA digestion and ligation protocols 1.3.1 Digestion Protocol For Plasmid Backbone Using EcoRI and PstI 1.3.2 Digestion Protocol For Plasmid pUC57 + Construction Using EcoRI and PstI 1.3.3 Ligation 1.3.4 Colony PCR 1.4 Biobricks protocols 2 Media and solutions protocols 2.1 LB broth for bacteria 2.1.1 LBA broth for bacteria 2.1.2 LB + Chloramphenicol broth for bacteria 2.2 SOC broth for bacteria 2.3 YP broth for yeast 2.3.1 YPD broth for yeast 2.3.2 YPRE broth for yeast 2.4 SD broth for yeast 2.5 LISORB solution for yeast transformation 2.6 40%PEG/LiAc/TE solution for yeast transformation 3 Yeast Induction protocol




General Protocols

Transformation protocols

Heat Shock Protocol for bacteria transformation

1. Take competent E.coli cells from –80°C freezer.
2. Turn on water bath to 42°C.
3. Put 100 ul of competent cells in an Eppendorf tube.
4. Keep tubes on ice.
5. Add 50 ng of circular DNA into E.coli cells. Incubate on ice for 10 minutes to thaw competent cells.
6. Put tube(s) with DNA and E.coli into water bath at 42°C for 45 seconds.
7. Put tubes back on ice for 2 minutes to reduce damage to the E.coli cells.
8. Add 1 ml of SOC Broth (with no antibiotic added). Incubate tubes for 1 hour at 37°C.
9. Spread about 100 ul of the resulting culture on LB plates (with Ampicillin added). Grow overnight.
10. Pick colonies about 12-16 hours later.

Yeast transformation

1. Prepare a 2 ml preculture in the selection medium of the strain to be transformed.
2. Inoculate 20 ml of YPD2% for each transformation, in order to get an OD600 = 1 next day.
   
   Where n is the number of divisions (generation time: 1´5 h for S. cerevisiae).
   From now, it is not necessary to work below sterility conditions. Once reached the OD600 = 1:
3. Centrifuge at 3000 rpm 5 min.
4. Wash with sterile water.
5. Resuspend in 30 ml of LISORB.
6. Shake at ambient temperature for 30 min.
7. Centrifuge at 3000 rpm 5 min and resuspend in 1 ml of LISORB. Transfer to an eppendorf tube.
8. Centrifuge at 3000 rpm 5 min.
9. Resuspend in 100 μl of LISORB for each transformation. Transfer 100 μL aliquots in different tubes for each transformation.
10. Add 7 μl of salmon sperm DNA + 1 μl of transforming DNA.
11. Incubate 10 min at ambient temperature.
12. Add 260 μl of 40%PEG/LiAc/TE. Mix well.
13. Incubate 1 h at 30°C.
14. Add 43 μl of DMSO and give a thermal shock of 5 minutes at 42°C.
15. Centrifuge at 3000 rpm 5 min.
16. Wash with 1 ml of sterile water.
17. Centrifuge at 3000 rpm 5 min.
18. Resuspend in 0´5 ml of water and plaque:
    -50 μl
    -Rest (centrifuge and decant leaving 50-100 μl).

DNA extraction and purification protocols

Mini-prep

1. Different cultures (each one with a different construction), which are growing in a selective media (LB + Ampicillin), get centrifuged at 4500g 5 min.
2. Supernatant is removed.
3. The cells can be washed (x2) with a saline solution (PBS) in order to remove impurities.
4. 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.
5. Transfer the suspension to an eppendorf tube.
6. Add 250 μL of Lysis Solution.
7. 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.
8. Add 350 μL of Neutralization Solution.
9. 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.
10. Centrifuge 10’ (max. rpm) in order to pellet cell debris and chromosomal DNA.
11. Transfer the supernatant (≈ 800 μL) to the spin column (pipetting to avoid carrying impurities).
    Important: DO NOT TRANSFERING THE PRECIPITATE!
12. Centrifuge 1’.
13. Flow-though liquid is removed.
14. Add 500 μL of Wash Solution (Solution stock has to be perfectly closed, it contains ethanol!).
15. Centrifuge ≈ 1’.
16. Flow-though liquid is removed.
17. 14, 15, 16 steps are repeated.
18. Centrifuge 1’ in order to eliminate residual Wash Solution.
19. The spin column is transferred into an eppendorf tube (the collection tube is eliminated).
20. 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!
21. Centrifuge ≈ 2’.
22. To increase the efficiency (≈ 20%) we can get the flow-though liquid and repeat the steps previously described (20 and 21).
23. The column is discarded and the solution which contains the purified plasmid can be stored in cold.

Protocol for Gel Extraction

1. Cut bands of interest from the agarose gel.
2. Add 300 uL of Solution L1 for each 100 mg of gel.
3. Incubate at 50ºC for 15 minutes.
4. Centrifugate in a 2 ml column at 12000xg for 1 minute.
5. Re-insert the spin column into the resaver tube and add 500 uL of Buffer L2.
6. Centrifugate 12000xg for 1 minute.
7. Discard the flow-through.
8. Centrifuge 12000xg for 1 minute.
9. Place the spin column into a new 1.5 mL microfuge tube.
10. Add 50 uL of mQ water.
11. Centrifuge 12000xg for 2 minutes.

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.
1. The digestion mixture is kept for 3 hours at 37 ° C
2. The mixture is kept for 20 minutes at 80ºC

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
1. 2 uL of EcoRI is added and incubated for 1 hour.
2. 2 uL of EcoRI is added and incubated for 1 hour.
3. 2 uL of PstI is added and incubated for 1 hour.
4. 2 uL of PstI is added and incubated for 1 hour.

Ligation

Plasmid DNA*		X uL
Insert DNA*		Y uL
10X ligase buffer		1 uL
T4 ligase		1 uL
mQ Water		8-(X+Y) uL
TOTAL		10 uL


OBSERVATION: The ratio that has to exist between the number of molecules of plasmid DNA and insert is 1:3 (the volumes depends on the concentration of DNAp and the insert).

Colony PCR

1. Each colony is taken from the petri dish and
   1. resuspended in 15 μl of mQ water in a eppendorf if we are working with bacteria.
   2. resuspended in 15 μl of NaOH 20 mM in a eppendorf if we are working with yeast.
2. Incubate for 15 minutes at room temperature.
3. 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
4. Once mixed, 4 ul of 10X TAQ polymerase solution is added.
5. 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
   72ºC 10 minutes
   4ºC Hold

Biobricks protocols

1. Digestion Protocol For Plasmid Backbone Using EcoRI and PstI
2. Digestion Protocol For Plasmid pUC57 + Construction Using EcoRI and PstI
3. Transformation Protocol Using Heat Shock
4. Ligation Protocol
5. Mini-preps. Purification protocol.
6. Protocol for Gel Extraction

Media and solutions protocols

LB broth for bacteria

Bacterial peptone		1% (p/v)
Yeast extract		0.5% (p/v)
NaCl		1% (p/v)

In order to obtain solid LB, add 15 g/L of bacteriologic agar.

LBA broth for bacteria

Add 1 mL of ampicilin 100mg/mL (of mQ water) in 1L of LB Broth

LB + Chloramphenicol broth for bacteria

Add 1 mL of Chloramphenicol 34mg/mL (of pure ethanol) in 1L of LB Broth

SOC broth for bacteria

Bacterial triptone		4g
Yeast extract		1g
NaCl 5M		0.4 mL
KCl 3M		0.167 mL
MgSO4		2.465 g
MgCl2		2.033 mL
Glucose		3.603 g
Distiled water		220 mL

Sterilization by filtration

YP broth for yeast

Bacterial peptone		2% (p/v)
Yeast extract		1% (p/v)

In order to obtain solid YP, add 2% of agar before sterilization.

YPD broth for yeast

Add x% (p/v) of dextrose or glucose (sterilized by filtration) in YP Broth after sterilization.

YPRE broth for yeast

Add 2% (p/v) of raffinose and 2% (v/v) of ethanol 100% in YP Broth after sterilization.

SD broth for yeast

Yeast nitrogen base w/o aminoacids and w/ amonium persulfate		0.67% (p/v)
Glucose		2% (p/v)


Aminoacids (leucine, metionine, histidine) and nucleotides (uracil) are added after sterilization in a final concentration of 20 mg/mL.
In order to obtain solid SD, add 2% (p/v) of agar before sterilization.


LISORB solution for yeast transformation

For 100 mL


LiAc 1M		10 mL
Sorbitol 2.4M		41.6 mL
TE 100X		1 mL

Bring to 100 ml of distilled H2O

40%PEG/LiAc/TE solution for yeast transformation

For 20 mL


LiAc 1M		2 mL
PEG 3500 50%		16 mL
TE 100X		0.2 mL

Bring to 20 ml of distilled H2O

Yeast Induction protocol

1. Colonies are picked from petri dish and suspended in suplemented SD media.
2. Incubate overnight
3. Measure OD.
4. Resuspend in YPD8%
5. Incubate overnight to 5 OD.
6. Resuspend in YPRE
7. Incubate for 8 to 24 hours
8. Add a final concentration of 0.3 mM of H2O2
9. Measure OD and fluorescence.