Protocols

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Needed supplies:

 0.5 m column, PVC 1 in. diameter, funnel at top, cap on bottom from:

 www.brewhaus.com

 Mixed bed de-ionization resin:

 Mixed Bed Standard Model Refill Kit Part OTG2-SDI-1

 Likely enough to make several hundred liters.

From:

http://www.portablewaterdi.com/refill-kits.php

 6 gal plastic bucket for storing product from:

http://www.ebrew.com/

 clamps, filter paper, plastic tube to fit.

 6-7 gal distilled H20, Krogers or other

Column prep and running:

Rinse column with distilled H20, close clamp so column is 1/2 filled with H20.
Add resin to H20 in container, mix well pour into column slowly until column is filled. Do not over fill funnel. Rinse column with 1 gal H20. Throw this out or use to rinse storage bucket.
Run the rest of H20 through column, at a trickle.
Put output into bucket.
Do NOT let column run dry.
At start, check the distilled H20 with a TDS meter (comes with resin), check the product occasionally.
Should take around 3-4 hrs.

Inoculate strain into Vogels agar media w/appropriate supplements
25 ml media in 250 ml flasks, two at least. Incubate 30C for at least 1 week
then move under lamp to induce max conidiation for at least a week; use as 2-3 week old cultures

Prep 2 x 200 ml bottles of 1M sorbitol ahead of time, autoclave and store at 4C

Conidia prep for electroporation

Add 30-40 ml of cold 1M Sorbitol to one flask and suspend conidia, transfer to second flask and repeat
Filter through sterile cheesecloth
Add 20-30 ml to one flask to capture more conidia, transfer to second flask and filter through cheesecloth
Rinse cheesecloth with 1M Sorbitol
Have ice bucket ready, move conidial suspension into 15 ml falcon tubes
Spin conidia at 3K RPM, resuspend in 5-10 ml 1M Sorbitol
Repeat this 1M Sorbitol wash 3 times
Finally resuspend conidia in minimal amount of 1M Sorbitol, usually around 1 ml
Take a 1:10 dilution of condia and count conidia in hemacytometer, use following website to calculate concentration:

http://www.changbioscience.com/cell/hemo.html

or: count cells in 1 square of 16 and cell conc. is
cell count x dilution x 25 x 104

adjust conidial suspension to 2 x 10(9)/ml

Media prep

Regeneration agar (100 ml)

50X Vogels          2 ml
Sorbitol          18.2 g
yeast extract       2 g
agar                   0.5 g

Plating media (90 ml)

50X Vogels        2 ml
yeast extract       2 g
agar                    1 g

Add 10X BdeS and hygromycin (50 mg/ml) to final conc of 300 ug/ml after cooling and plate

Recovery media

50X Vogels       2 ml
Sorbitol            18.2 g
yeast extract         2 g

500X his (for use in above solutions with his auxotrophs)
2.5 g L-histidine in 25 ml ddH2O
use 2 ml/l

Electroporation conditions

2 mm cuvette, place cuvettes on ice prior to use
place microfuge tubes on ice, add plasmid DNA, various concentrations, use at least 0.5 ug and add 40-50 ul conidia, sit 5 min
add conidia/DNA suspension to cuvette and zap with following conditions

1.5 kV, 25 uF, 600 ohms

Add 1 ml of recovery media to cuvette and transfer to microfuge tube; incubate at 30C 1 hr to recover
Add each sample to 8-9 ml of regeneration agar in 15 ml falcon tube maintained at ~55C and plate; incubate at 30C for 4-5 days
Pick colonies to Vogels sucrose + 200 ug/ml hygromycin tubes (also do original strain as control) and incubate 30C until they conidiate

Final result: happy transformants (74 is the parental untransformed strain) media is Vogels sucrose with 200 ug/ml hygromycin

Unlabeled plate - no DNA control, labeled plates transformed with pCSN44

PCR of the above two transformants with primers to amplify hygR selection marker

       1             2           3           4          5

Lane 1 1 kb ladder
Lane 2 no DNA control
Lane 3 74A (parental strain)
Lane 4 T1
Lane 5 T2

band indicates hygromycin resistance from transforming plasmid pCSN44

Small prep of cells

Make 0.1 M CaCl2 and 0.1 M CaCl2 + 15% glycerol and store at 4C

Begin with fairly fresh streak of E coli (< 1 month)
Inoculate into 5 ml SOB
Shake overnight at 37C
Add to 50 ml SOB
Shake at 37C until OD is right, 3-4 hrs
Distribute into 4 15 ml tubes @ 10 ml/tube
Incubate on ice 20 min
Spin in tabletop 2 min
Resuspend in 4 ml 0.1 M CaCl2 20 min and
recentrifuge
Resuspend in 2 ml 0.1 M CaCl2, 15% glycerol
Use immediately and stock the rest in 1.5 ml eppendorfs (400 ul/tube)
Quick freeze in dry ice if available, store at -20C


Transformation of DNA samples
Add DNA to 200 ul of competent cells
Incubate 30 min on ice
Heat shock 90 sec at 42C
Ice for 2 min
Add 800 ul of SOB (or SOC)
45 min at 37C
Take 100 ul aliquot and plate, spin rest down and plate to have different concentrations
as transformation efficiency may be variable

TSS transformation of E. coli

2X TSS
20% PEG 4000
10% DMSO
40 mM MgSO4
store at 4C

Inoculate colony of E. coli into 5 ml  LB, grow overnight at 37C
Inoculate 1 ml of above into fresh flask of 50 ml SOB, shake until OD = 0.3-0.5
Place cells on ice for 10 min
Spin down to 1/10th volume (5 ml) and ice, use 1 ml original culture per sample
Mix cells 1:1 with cold 2X TSS and ice.
Add DNA to eppendorf on ice
Add 100 ul cell mixture to DNA
Incubate on ice 30-60 min
Add 0.9 ml of SOC to cells, shake at 37C for 1 hr
Plate on antibiotic media (LM + antibiotic)

adapted from PNAS 86:2172 (Chung, Niemela, Miller)

Electroporation of Saccharomyces cerevesiae

1. Inoculate 50ml YEPD with a colony and grow with shaking at 30C until early stationary (~0.6-2E8 cells/ml).
2. Harvest in a 15 ml sterile tube in clinical centrifuge spin at top speed, 4C, 2' and keep cells on ice throughout the procedure.
3. Wash cells with 40ml ICE-COLD sterile dH2O, pellet, 4C, 2'.
4. Repeat wash with 20ml sterile dH2O (ice-cold).
5. Resuspend cells in 5ml 1M Sorbitol (ice-cold) pellet, 4C, 2'.
6. Resuspend cells with 150µl 1M Sorbitol (ice-cold) - KEEP ON ICE!
7. Mix 40 µl of yeast suspension with <5 µl DNA (~5 µg) in a prechilled electroporation cuvette (0.2 cm). Tap contents to the bottom, making sure that the sample is in contact with both sides of the aluminum cuvette.
8. Give one pulse: V= 1.5kV, 25 µF, 200 Ohms. Time should be ~4-5".
9. Immediately add 1 ml 1M Sorbitol (ice-cold) and transfer with a sterile pasteur pipette to a sterile Eppendorf tube.
10. Spread onto selective plates.

Store all primers at 1 ug/ul in ddH2O for use in reactions below.

Primer calculations:
http://www.promega.com/biomath/Default.htm

20 mer @ 1 ug/ul = 151 pmol
25 mer @ 1 ug/ul = 121 pmol

Program 14 > 12 > 11
94C for 6 min
followed by 30 cycles of:
94C, 1 min
55C, 1 min
72C, 2 min
12C until refrigerated

Program 20
94C for 6 min
followed by 30 cycles of:
94C, 1 min
60C, 1 min
72C, 2 min
12C until refrigerated

master mix for 4 rxns (50 ul each rxn):
10X ThermoPol buffer   20 ul
10 mM dNTPs                4 ul
primer 1                         4 ul
primer 2                         4 ul
ddH2O                           165 ul
Taq                                1 ul

LB
0.5% yeast extract
0.5% NaCl
1% tryptone

SOB
200 ml
1 g yeast extract
4 g tryptone
10 mMNaCl
0.2 ml of 1M KCl
0.2 ml 1M MgCl2 (1mM final)
0.2 ml 1M MgSO4 (1 mM final)
for SOC add 1/100 2M glucose

LM (1 liter)
10 g tryptone
5 g yeast extract
0.58 g NaCl (1 mM final)
2.46 g MgSO4 (1 mM final)
pH 6.8-7.0

TB
1.2% tryptone
2.4% yeast extract
0.4% glycerol

1X M9 salts
2mM MgSO4
0.1mM CaCl2
0.4% carbon source (e.g. glycerol, glucose, etc.)
In sterile H2O

5X M9 salts contains:
64 g Na2HPO4.7H2O (for 250 ml; 16 g)
15 g KH2PO4 (3.75 g)
2.5 g NaCl (0.625 g)
5 g NH4Cl (1.25 g)
dissolved in deionized water to a final volume of 1 L

YPD
1 % yeast extract
2 % peptone
2% dextrose

SD-Ura (100 ml)
2.67 g dropout base with glucose (USB #D9500)
0.2 g dropout mix minus Ura (USB #D9535)
1.5 g agar
pH 5.8 with 1M NaOH

Vogels sucrose minimal
200 ml
4 ml 50X Vogels
3 g sucrose

50X Vogels stock (for 250 ml)
Na2 citrate 2H2O    37.5   g
KH2PO4                  62.5   g
NH4NO3                 25      g  
MgSO4 7H2O            2.5   g
CaCl2 2H2O              1.25 g
Biotin stock               1.25 ml
Trace elements          1.25 ml (from aquarium store is sufficient)

4X Westergaards (per liter)
KNO3                            8 g
KH2PO4                        8 g
MgSO4 7H2O               4 g
Biotin                            8 ml
Trace + NaCl + CaCl2  8 ml

10X BdeS (100 ml)
10% sorbose       10 g
0.5% glucose     0.5 g
0.5% fructose    0.5 g
autoclave, add CHCl3

amino acid supplements add 1/100 of aa stock
vitamin supplements add 1/1000 of vitamin stock
from FGSC; www.fgsc.net
stock    
p-aminobenzoic acid    0.4   mg/ml        0. 5 ml
choline chloride           2.0                  1.5
inositol                         5.0                  1.0
nicotinamide                5.0                  0.2
Ca-pantothenate          1.0                  1.0
pyridoxine HCl           1.0                  1.0
thiamine                      1.0                  1.0
L-arginine                 40.0                  1.25
L-histidine HCl         25.0                  2.0
DL-homoserine         10.0                  2.0
indole                          1.0                  2.0
L-leucine                     5.0                  4.0
L-lysine                     20.0                  2.5
L-methionine             10.0                  5.0
L-phenylalanine         10.0                  2.0
L-proline                    10.0                  5.0
sulfanilamide               3.4                  1.0
L-threonine                  5.0                  2.0
L-arginine                  20.0                  2.5
     + L-lysine             40.0                  1.0
L-isoleucine                3.0                   3.0
     + L-valine              7.0                   1.0

Individual supplements are dissolved in water and stored at 5∞C over chloroform.

Adenosine, adenine sulphate, adenylic acid, uracil, and tyrosine are not sufficiently
soluble to allow the use of concentrated stock solutions. Adenosine is used at 0.5 mg/ml, uracil at 1.0 mg/ml, and tyrosine at 0.4 mg/ml.

hygromycin B
stock at 50 mg/ml in H2O; use at 2-300 ug/ml

cycloheximide
stock at 10 mg/ml; use at 10 ug/ml

ignite
stock at 600 mg/ml; use at 400 ug/ml


E coli supplements
amp 50 ug/ml from 25 mg/ml stock, filter sterilized (200 ul/ 100 ml media)

x gal use 1 ul/ml media
make stock at 20 mg/ml (200 mg in 10 ml DMF)

IPTG use at 100 uM, (1 ul/ml media)
make stock (100 mM) 238 mg/10 ml ddH2O