Team:Valencia Biocampus/Protocols

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Protocols

Heat Shock Protocol

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 LB (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.

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.

Digestion

Ligation

Yeast transformation

1. Prepare a 2 ml preculture in the selection medium of the strain to be transformed.
2. 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).
   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).

Colony PCR

1. Each colony is taken from the petri dish and resuspended in 15 μl of NaOH 20 mM in a eppendorf.
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

Colony PCR

1) Each colony is taken from the petri dish and resuspended in 15 μl of NaOH 20 mM.

2) Turn on water bath to 42°C.