IGEM:University of Chicago/2009/Protocols

Media/Solutions

MOPS Buffered Minimal media

• MOPS Buffered Minimal Media

'Protocol: This protocol outlines the formulation of MOPS buffered minimal media. This media should be used for e.coli growth under paraoxon induction. MOPS buffered minimal media was formulated using the following ingredients

• adapted from Neinhardt, et al. J. Bact. Sept. 1974. Vol. 119, No. 3. p. 736-747

• Final concentration (10X)

1. 40 mM MOPS
2. 4 mM tricine
3. 10 uM FeSO4
4. 9.5 mM NH4Cl
5. 0.5 uM CaCl2
6. 0.521 mM MgCl2
7. 50 mM NaCl
8. 0.276 mM K2SO4
9. 3 x 10-9 M Ammonium Paramolybdate
10. 4 x 10-7 M Boric Acid
11. 3 x 10-8 M CoCl2
12. 1 x 10-8M CuSO4
13. 8 x 10-8 M MnCl2
14. 1 x 10-8 M ZnSO4
15. 1.32 mM K2PO4 (Phosphorus source)

• Dilute to 1X in sterile ddH2O
• Carbon sources will be added separately ( 1 mM, or 0.5% glycerol added separately).

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

1. Melt agar (microwave ~4min in Annette’s room, tighten cap)
2. Let cool 10-15min (not too cool)
3. Add in Glucose and G418 to 2X YEP bottle, mix
4. Add 3) to cooled agar, mix
5. Spray 95% EtOH to the surface to get rid of air bubbles
6. Pour immediately onto plates, let cool
7. 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.

Glycerol stocks

Freezing down yeast strains

• From Plate

1. Mix 1ml 30-50% grycerol (sterile)* and 1ml YPD in cryotube
2. Scoop cells from plate (half-pea size), put in the tube
3. Vortex, store at -80ºC

• From culture

1. Mix 1ml 30-50% grycerol (sterile)* and 1ml culture in cryotube
2. Vortex, store at -80ºC

• dilute glycerol in dH2O, filter sterilize

Freezing down bacterial strains

• Same as freezing down yeast from culture.

Transformations

Pryciak Lab LiAc Yeast Transformation

• Standard Small Scale Protocol

• 50mL cµlture = 20 transformations
• 10mL cµlture = 4 transformations

1. Set up stocks of strains 3-4mL. Grow 30C overnight.
2. In morning, inocµlate 50mL (or 10mL) YPD with 1:50 dilution of stock culture.
3. Incubate cultures 3-4 hours @ 30C (OD600 of 0.5-1.0 = 1-2 X 107)
4. Transfer cultures to falcon tubes.
5. Spin down cells for 2-3 minutes @ 2-3,000 rpm at room temp. Decant.
6. Resuspend in 5mL (2mL) sterile TE. Spin 2-3 min @ 2-3,000 rpm. Decant.
7. Resuspend in 5mL (2mL) LiAc mix. Spin 2 min @ 2-3,000 rpm. Decant.
8. Resuspend in 500µl (100µl) LiAc mix.
9. Add 1.5µl boiled* ssSalmon Sperm DNA (10mg/ml) to eppendorf tube

before using: boil 10m in bead bath, then ice.

1. Add 0.1-5µg DNA to tube (1-2µl miniprep DNA is usually enough)
2. Add 25µl cell suspension to each tube, mix.
3. Add 200µl PEG mix to each tube then vortex.
4. Incubate at least 30 minutes @ 30C.
5. Heat shock 20 minutes @ 42C.
6. To pellet spin 30 seconds.
7. Resuspend in SOS (usually 20µl for plasmids, 200µl for integration).
8. Plate on selective media (streak out 10µl for plasmids, 20µl +180µl for integration).
9. 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#
• DH5alphacompetent cells.......100µl/rxn, 200 or 400µl/tube at -80ºC
• Culture tubes..................sample#

• Check water bath temp at 42ºC

1. Thaw competent cells on ice
2. Add DNA to tube, put on ice
3. Add 100µl of cells to each tube, pipet up and down to mix
4. Put on ice for 15-30min
5. Heat shock for 45sec at 42ºC
6. Put on ice for 2min
7. Add 500µl of 2XYT to tube
8. Transfer entire vol. into culture tube
9. Incubate at 37C for 1h
10. 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.

DNA prep and purification

Gel-purification (Zymo)

Zymoclean Gel DNA Recovery Kit

• Before starting, add 24mL 100% ethanol to the 6ml DNA Was Buffer concentrate.

• Materials
• ADB Buffer
• Wash buffer
• Sterile ddH2O or TE buffer
• Zymo-Spin I Column

• Method

1. Excise the DNA fragment from agarose gel using sterile razor blade or scalpel and transfer to a 1.5mL microcentrifuge tube
2. Add 3 volumnes of ADB buffer to each volume of agarose excised from the gel

Note: DO NOT use the UV visualizer for this. Also, this doesn't have to be terribly accurate. We usually put in about 150μL for our gel samples, but it depends on how much gel you cut out. In general, try to cut off as much of the agarose as possible without cutting out any of the DNA.

1. Incubate at 37-55C for 5-10 minutes until the gel slice is completely dissolved.
2. Transffer the melted agarose solution to a Zymo-spin I Column in a Collection Tube.
3. Centrifuge at >10,000rpm for 30-60s. Discard the flow-through.
4. Add 200μL of Wash Buffer to the columns and centrifuge at >10,000 rpm for 30 seconds. Discard the flow-through. Repeat.
5. Add 6-10 (we usually use 20μL) of ddH2O or TE buffer directly to the column matrix. Place column into a 1.5mL tube and centrifuge >10,000rpm for 30-60 seconds to elute DNA.
6. DNA is now ready for use!
7. Incubate

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.