45% (w/v) Sterile Glycerol
450 grams enzyme grade glycerol per liter
Autoclave and store at room temperature.
T low E Buffer
1M Tris-HCl (pH 8.0) 10 mls
0.5 M EDTA (pH 8.0) 0.2 mls
DEPC treated H2O 990 mls
1000
Autoclave and store at room temperature.
Carbenicillin stock solution
1 gram of carbenicillin in 10 mls of sterile water.
Sterile filter with a 0.2 micron filter.
Store frozen at -20°C.
LB with 100 µg/ml carbenicillin
Add 1 ml of carbenicillin stock solution to 1 liter of LB.
Make fresh.
3M Sodium Acetate pH=6.0
Prepare 3M sodium acetate
408.24 grams sodium acetate (tri-hydrate) per liter
Prepare 3M acetic acid
172.4 ml per liter
Titrate the pH of the 3M sodium acetate solution to pH 6.0 with the 3M acetic acid solution.
Filter sterilize using a 0.2 micron filter.
Store at room temperature.
Ethanol/acetate mix
Ethanol (100%) 950 ml
Sodium acetate pH=6.0 50 ml
1000 ml 3X SSC
DEPC H2O 42.5ml
20X SSC 7.5ml
50ml 70% Ethanol
Ethanol (100%) 350 ml
DEPC H2O 150 ml
500 ml
2.1 EST clone growth
1. Incubate sealed master plates over night at 37°C.
Most suppliers provide low density bacterial cultures. Replicating directly from these dilute stocks frequently results in non-growth in the secondary culture. If making template from a plate that has previously been cultured to high density before freezing, this initial growth step should not be used, as it will reduce the viability of the cultures.
2. Prepare sets of standard 96 well round (U) bottom plates by labeling all plates and placing 100 µl of LB broth containing 100ug/ml carbenicillin in each well. These plates will be used as working copies.
To preserve the master set of plates, it is useful to make replicate copies of the master plate to serve as working copies when the master plate is first replicated. Check to insure that the EST clones are in a vector conferring ampicillin resistance, as is common with human IMAGE clones.,
4. Spin the master plates briefly, two minutes, at 1000 rpm in a horizontal microtiter plate rotor to remove condensation and droplets from the seals before opening.
Bacterial culture fluid on the sealers can easily be transferred from one well of the plate to others, cross-contaminating the stocks.
5. Partially fill a container with 100% alcohol. Dip the 96 pin-replicating tool in the alcohol. Remove from the alcohol bath and then fla me the pins.
6. Allow the inoculation block to cool briefly, then dip the replicating tool in the master plate, and then into the daughter plate. Repeat as necessary for each plate that you need to inoculate.
It is useful to color the plate corner near the A-1 well of all master and daughter plates with a marker pen before beginning the replication process, to reduce mistakes in relative orientation of the plates. The suggested plates have a notch at this corner as well.
7. Place the inoculated LB plates with lids on into a one gallon "zip-lock" bag containing a moistened paper towel and grow overnight at 37°C.
Many 37°C incubators tend to dry out microtiter plate cultures. Placing the plates in a highly humidified bag avoids this problem.
3. Fill deep well plates with 1ml of Superbroth (100ug/ml carbenicillin) per well. These plates will serve as the source of culture for template preparation.
8. Using the replicating tool, inoculate the deep well plates directly from the freshly grown LB plates.
9. Cover the openings of the deep well plates with Qiagen Airpore Tape Sheets and place the plastic lid over the sheet. Place the plates in a 37°C shaker incubator at 200 RPM for twenty-four hours.
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