IGEM:University of Chicago/2009/Protocols: Difference between revisions
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*H2O--'''6.6mL of H2O''' | *H2O--'''6.6mL of H2O''' | ||
*6.5mM CaCl--'''65µl of 1M CaCl''' | *6.5mM CaCl--'''65µl of 1M CaCl''' | ||
=Plates= | |||
*YPD+Kan plate (Kron Lab) | |||
*'''Ingredients'''.....Amount..........Final [] | |||
*2X YEP................300ml.................1X | |||
*Agar (4%).............300ml.................2% | |||
*Glucose (40%).........30ml..................2% | |||
*G418 (50mg/ml stock)..2.4ml.................200µg/ml | |||
# Melt agar (microwave ~4min in Annette’s room, tighten cap) | |||
# Let cool 10-15min (not too cool) | |||
# Add in Glucose and G418 to 2X YEP bottle, mix | |||
# Add 3) to cooled agar, mix | |||
# Spray 95% EtOH to the surface to get rid of air bubbles | |||
# Pour immediately onto plates, let cool | |||
# When solidify, mark plates (three blue lines), put back in a bag, and store at 4ºC | |||
*2X YEP, 4% Agar, 40% glucose, plates, and 95% EtOH bottle are all in Annette’s room. | |||
*G418 (Geneticin) stock is in Kron Lab 4ºC fridge for hazardous reagents. | |||
*Kanr gene in the Longtine vectors confers resistance to G418 (=Geneticin) in yeast. We still call them “Kan” plates although they really are G418 plates. | |||
=Transformations= | =Transformations= |
Revision as of 12:02, 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
Plates
- YPD+Kan plate (Kron Lab)
- Ingredients.....Amount..........Final []
- 2X YEP................300ml.................1X
- Agar (4%).............300ml.................2%
- Glucose (40%).........30ml..................2%
- G418 (50mg/ml stock)..2.4ml.................200µg/ml
- Melt agar (microwave ~4min in Annette’s room, tighten cap)
- Let cool 10-15min (not too cool)
- Add in Glucose and G418 to 2X YEP bottle, mix
- Add 3) to cooled agar, mix
- Spray 95% EtOH to the surface to get rid of air bubbles
- Pour immediately onto plates, let cool
- When solidify, mark plates (three blue lines), put back in a bag, and store at 4ºC
- 2X YEP, 4% Agar, 40% glucose, plates, and 95% EtOH bottle are all in Annette’s room.
- G418 (Geneticin) stock is in Kron Lab 4ºC fridge for hazardous reagents.
- Kanr gene in the Longtine vectors confers resistance to G418 (=Geneticin) in yeast. We still call them “Kan” plates although they really are G418 plates.
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
- DH5alpha transformation (Kron Lab)
- Epp. tubes (sterile)...........sample#
- DH5alphacompetent cells.......100µl/rxn, 200 or 400µl/tube at -80ºC
- Culture tubes..................sample#
- Check water bath temp at 42ºC
- Thaw competent cells on ice
- Add DNA to tube, put on ice
- Add 100µl of cells to each tube, pipet up and down to mix
- Put on ice for 15-30min
- Heat shock for 45sec at 42ºC
- Put on ice for 2min
- Add 500µl of 2XYT to tube
- Transfer entire vol. into culture tube
- Incubate at 37C for 1h
- Plate out onto LB+Amp or other appropriate selection plates (10µl + 200µl 2XYT / rest ~500µl)
1#Incubate at 37C overnight
- competent cells are in -80ºC freezer#1 in the heavy equipment room, Shelf#1 (stocks & bugs), metal rack on the right, 3rd drawer. There are two boxes of competent cells (400µl/tube and 200µl/tube).
- 2XYT is in Annette’s room.
- 37ºC shaker is in the heavy equipment room.
PCR
AccuSure Polymerase
- Set up two solutions
- Solution 1
10X buffer--2 μL dNTPs (10mM stock)--0.5μL template--.0.25 μL forward primer--2 μL reverse primer--2 μL polymerase--1 μL Total: 10 μL
- To this solution add 10 μL of solution 2
- Solution 2
This sets up a magnesium gradient. When testing for optimal conditions, 4 different concentrations of MgCl may be tried. MgCl volumes are for a 20mM stock solution. A: 0 μL MgCl, 10 μL ddH2O B: 1 μL MgCl, 9 μL ddH2O C: 2 μL MgCl, 8 μL ddH2O D: 4 μL MgCl, 6 μL ddH2O
- For the most part using using water worked fine, as the 10X buffer already contains some magnesium.
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.