Team:Valencia Biocampus/Protocols
From 2012.igem.org
(Difference between revisions)
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<ol>inoculum vol.= (final OD)/(preculture OD)×(final vol.)/2^n | <ol>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).</ol> | Where n is the number of divisions (generation time: 1´5 h for S. cerevisiae).</ol> | ||
- | + | From now, it is not necessary to work below sterility conditions. | |
- | <li> | + | Once reached the OD600 = 1: |
- | <li> | + | <li>Centrifuge at 3000 rpm 5 min. |
- | <li>Add | + | <li>Wash with sterile water. |
- | <li> | + | <li>Resuspend in 30 ml of LISORB. |
- | <li> | + | <li>Shake at ambient temperature for 30 min. |
- | <li> | + | <li>Centrifuge at 3000 rpm 5 min and resuspend in 1 ml of LISORB. Transfer to an eppendorf tube. |
- | <li> | + | <li>Centrifuge at 3000 rpm 5 min. |
- | <li> | + | <li>Resuspend in 100 μl of LISORB for each transformation. Transfer 100 μL aliquots in different tubes for each transformation. |
+ | <li>Add 7 μl of salmon sperm DNA + 1 μl of transforming DNA. | ||
+ | <li>Incubate 10 min at ambient temperature. | ||
+ | <li>Add 260 μl of 40%PEG/LiAc/TE. Mix well. | ||
+ | <li>Incubate 1 h at 30°C. | ||
+ | <li>Add 43 μl of DMSO and give a thermal shock of 5 minutes at 42°C. | ||
+ | <li>Centrifuge at 3000 rpm 5 min. | ||
+ | <li>Wash with 1 ml of sterile water. | ||
+ | <li>Centrifuge at 3000 rpm 5 min. | ||
+ | <li>Resuspend in 0´5 ml of water and plaque: | ||
+ | <ol>-50 μl | ||
+ | -Rest (centrifuge and decant leaving 50-100 μl).</ol> | ||
+ | |||
</ol> | </ol> | ||
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Revision as of 08:46, 22 September 2012
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.
- 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.
- 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).
Heat Shock Protocol
Mini-prep
Digestion
Ligation
Yeast transformation
1-
2-
3- 4-
5-
6-
7-
8- 9-
10-
11-
12-
13-
14-
15- 16-
17-
18-
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:
-
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.