IGEM:University of Chicago/2009/Protocols: Difference between revisions
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(New page: =Media/Solutions= ==Solutions for LiAc Yeast Transformations== ===LiAc Mix (Store RT, Sterile filter), '''500mL'''=== *H2O--'''400mL''' *100mM LiAc--'''50mL of 1M LiAc''' *TE (10mM Tris+1...) |
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===AccuSure Polymerase=== | ===AccuSure Polymerase=== | ||
===Taq Polymerase/beads=== | ===Taq Polymerase/beads=== | ||
*illustra PuReTaq Ready to Go PCR beads (from GE Healthcare) | |||
*Supplied by Chris Schonbaum | |||
*Each bead is designed for use is a 25μL reaction. When resuspended, each reaction will contain 1.5mM MgCl2. | |||
*Each bead also contains (after final volume) | |||
200μM each dNTP in 10mM Tris-HCl (pH 9 at room temperature) | |||
50μM KCl | |||
*For each bead add | |||
5-25 pmol forward primer | |||
5-25pmol reverse primer | |||
Template DNA (50pg for plasmid DNA, 50ng for complex template such as genomic DNA. Avoid amounts >1μg) | |||
*Snap caps (provided) onto tubes. Mix by gently flicking tubes. Vortex then centrifuge for a few seconds. The reaction is fully dissoluved and mixed when it appears clear. | |||
*For our 50μL reactions we used | |||
2 beads | |||
5μL of each primer (5μM stock, final amount is 25pmol) | |||
.7μL template (plasmid, 30μM) | |||
37.3 μL ddH2O | |||
*Half all amounts for 25μL reactions. | |||
*For cell-culture PCR, we replaced template DNA with a blot of cell culture. |
Revision as of 10:13, 29 July 2009
Media/Solutions
Solutions for LiAc Yeast Transformations
LiAc Mix (Store RT, Sterile filter), 500mL
- H2O--400mL
- 100mM LiAc--50mL of 1M LiAc
- TE (10mM Tris+1mM EDTA, pH8)-- 50mL of 10X TE
PEG Mix (Store RT, Sterile filter), 10mL
- 40% PEG mw 3350--8mL of 50% PEG Sterile filtered
- 100mM LiAc--1mL of 1M LiAc
- TE--1mL of 10X TE
SOS (make fresh), 10mL
- 33%YEP--3.4mL of YEP(or YPD)
- H2O--6.6mL of H2O
- 6.5mM CaCl--65µl of 1M CaCl
Yeast transformations
Pryciak Lab LiAc Yeast Transformation
- Standard Small Scale Protocol
- 50mL cµlture = 20 transformations
- 10mL cµlture = 4 transformations
- Set up stocks of strains 3-4mL. Grow 30C overnight.
- In morning, inocµlate 50mL (or 10mL) YPD with 1:50 dilution of stock culture.
- Incubate cultures 3-4 hours @ 30C (OD600 of 0.5-1.0 = 1-2 X 107)
- Transfer cultures to falcon tubes.
- Spin down cells for 2-3 minutes @ 2-3,000 rpm at room temp. Decant.
- Resuspend in 5mL (2mL) sterile TE. Spin 2-3 min @ 2-3,000 rpm. Decant.
- Resuspend in 5mL (2mL) LiAc mix. Spin 2 min @ 2-3,000 rpm. Decant.
- Resuspend in 500µl (100µl) LiAc mix.
- Add 1.5µl boiled* ssSalmon Sperm DNA (10mg/ml) to eppendorf tube
before using: boil 10m in bead bath, then ice.
- Add 0.1-5µg DNA to tube (1-2µl miniprep DNA is usually enough)
- Add 25µl cell suspension to each tube, mix.
- Add 200µl PEG mix to each tube then vortex.
- Incubate at least 30 minutes @ 30C.
- Heat shock 20 minutes @ 42C.
- To pellet spin 30 seconds.
- Resuspend in SOS (usually 20µl for plasmids, 200µl for integration).
- Plate on selective media (streak out 10µl for plasmids, 20µl +180µl for integration).
- Grow 30C 2-3 days.
Note: cells can be frozen at step 8 in TE + 15% glycerol and stored @ -70C. Thaw slowly, spin down and resuspend in LiAc. However this significantly reduces competence.
E. coli transformation
PCR
AccuSure Polymerase
Taq Polymerase/beads
- illustra PuReTaq Ready to Go PCR beads (from GE Healthcare)
- Supplied by Chris Schonbaum
- Each bead is designed for use is a 25μL reaction. When resuspended, each reaction will contain 1.5mM MgCl2.
- Each bead also contains (after final volume)
200μM each dNTP in 10mM Tris-HCl (pH 9 at room temperature) 50μM KCl
- For each bead add
5-25 pmol forward primer 5-25pmol reverse primer Template DNA (50pg for plasmid DNA, 50ng for complex template such as genomic DNA. Avoid amounts >1μg)
- Snap caps (provided) onto tubes. Mix by gently flicking tubes. Vortex then centrifuge for a few seconds. The reaction is fully dissoluved and mixed when it appears clear.
- For our 50μL reactions we used
2 beads 5μL of each primer (5μM stock, final amount is 25pmol) .7μL template (plasmid, 30μM)
37.3 μL ddH2O
- Half all amounts for 25μL reactions.
- For cell-culture PCR, we replaced template DNA with a blot of cell culture.