[Arabidopsis] Semi Solid Attempt 2

Holt, Ben F. III via arab-gen%40net.bio.net (by benholt from ou.edu)
Sat Apr 4 18:00:48 EST 2009


Hi everyone,

Apparently there were problems with the last attachment - I went back and checked the message I sent and the attachment was there, so I conclude that the listserv is either blocking these in general or just didn't like the one I sent.

Anyway, I have attached it here again and I have pasted the two protocols below - sorry, but the pasted protocols below are not very pretty. I had to choose between making them look nice or watching the Final Four.

Go Heels!

Ben


Additional Protocols

Note: Carbenicillin reduces satellite
formation.

Caution: Make sure that the rotor was set
at room temperature for at least 2 h before
adding the bottles. Rotors at 4°C will
cause solidification of agar.

5.1 Expansion of Plasmid cDNA Libraries
Semi-solid amplification of primary cDNA transformants minimizes representational
biases that can occur during the expansion of plasmid cDNA libraries (25). Life
Technologies has further modified this protocol such that the amplification is done at
30°C, helping to stabilize unstable clones (26).

Caution: Bottles of semi-solid agar containing suspended colonies must be handled
gently and incubated without disturbance. Rough handling or bumping of the
incubator will cause micro colonies to fall out of solution. Incubators that have fans
can cause colonies to fall out of solution.

1.
Prepare 2.0 L of 2X LB. Remove 175 ml of the 2X LB to make the 2X LB
Glycerol.
2.
Place a large stir bar and 1.35 g SeaPrep® agarose into each of four 500-ml
autoclavable bottles. Place bottles on stir plates. With the stir plate turned on,
add 450 ml of 2X LB to each bottle (avoid the formation of large clumps of
agarose).
3.
Autoclave these bottles of 2X LB agarose for 30 min.
4.
Cool bottles in 37°C water bath for ~2 h until the medium reaches 37°C.
5.
After the medium reaches 37°C, add carbenicillin to 50 "g/ml (preferred
antibiotic) or add ampicillin to 200 "g/ml. Mix on stir plate.
6.
To each bottle add 4 %&105 to 6 %&105 primary cDNA transformants (colonies
from original library), and mix thoroughly on a stir plate for 2 min. Tighten caps.
7.
Place bottles in an ice-water bath (0°C) such that the water in the bath is at the
same level as the medium in the bottle. Incubate for 1 h in the ice bath.
8.
Gently remove bottles from the ice bath and wipe the water off the outside of
the bottles. Loosen bottle caps. Place these bottles in a gravity flow incubator
set at 30°C.
9.
Incubate these bottles for 40-45 h without disturbance. Place a centrifuge rotor
at room temperature the morning of harvest.
10.
Pour contents of bottles into centrifuge bottles and centrifuge at 10,400 * g for
20 min at room temperature
11.
Pour off the supernatant.
12.
Resuspend the cells in a total of 100 ml 2X LB Glycerol (12.5%). Remove two
100-"l aliquots for plating, further analysis, and colony estimate. Cells can be
filtered through sterile cheesecloth to remove agarose clumps if present.
13.
Subdivide the cells into ~10-ml aliquots and store at -70°C. It is also useful to
make a number of 1-ml and 100-"l aliquots.
14.
Frozen cells can be used to prepare DNA for GENETRAPPER(&experiments, for
plating to screen, or can be further amplified in liquid at 30°C to obtain DNA.
Use 2.5 %&109 cells/100 ml growth medium for further expansion of the library.
32




Media
2X LB
Component Amount/liter

5

Bacto-Tryptone................................................................. 20 g


Bacto-Yeast Extract.......................................................... 10 g
NaCl ................................................................................. 10 g


2X LB Glycerol (12.5%)
Component Amount/200 ml


2X LB............................................................................ 175 ml
Glycerol (100%)...............................................................25 ml


Filter-sterilize and store for up to 2 months at room temperature.




Amplifying the pCMV-Script® cDNA Library

The primary library may now be plated and screened; however,
amplification of the library is desirable to produce a large and stable
quantity of the library. Do not perform more than one round of
amplification, as slower-growing clones may be significantly
underrepresented.

Stratagene recommends amplifying plasmid libraries in 500-ml bottles of 2*
LB agarose§ using the semi-solid amplification method.4, 5 The libraries are
amplified in suspension which allows for three-dimensional, uniform colony
growth. This reduces the potential for under-representation of particular
clones due to the overgrowth of some colonies during the expansion process
that accompanies direct plating methods.

Note
Each 500-ml bottle of 2* LB agarose can accommodate ~5 * 105
primary cfu. To amplify a library of 1 * 106 primary cfu, two
bottles are necessary.

Note
Stratagene has determined that the use of SeaPrep® agarose is
critical to ensure performance (See Additional Materials
Required).

1.
On a heated stir plate, using a large stirbar, combine 1.35g of SeaPrep
agarose with 450 ml of 2* LB§ in each 500-ml autoclavable bottle.
Heat and stir until the agarose is in solution.
2.
Autoclave the bottles and stir bars for 30 minutes.
3.
Allow the bottles to cool to 37°C in a water bath (~1 hour).
4.
Transfer the bottles to a stir plate and add 50 *g/ml of kanamycin.
5.
Add up to 5 * 105 cfu/bottle of primary library and stir for several
minutes.
Note
The bottles must be handled very carefully at this stage.
Avoid swirling or bumping the bottles as this may cause the
microcolonies to fall out of suspension. The bottles must be
incubated without disturbance or representation of the
amplified library may be compromised.

6.
Tighten the bottle caps and incubate the bottles for 1 hour in an ice
water bath (0°C). The water level in the ice bath should be even with
the media level in the bottle.
7.
Carefully remove the bottles from the ice bath and gently dry the
bottles. Loosen the bottle caps and incubate for 40-45 hours at 30°C.
(Incubation at 30°C reduces under-representation of slower growing
clones.)
§ See Preparation of Media and Reagents.

pCMV-Script® XR cDNA Library Construction Kit


8.
Pour the contents of the bottles into sterile 250-ml centrifuge bottles
and spin at 10,000 * g for 20 minutes at room temperature.
(Equilibrate the rotor to room temperature several hours prior to
centrifugation. Using rotors stored at 4°C will cause the agar to
solidify.)
9.
Remove the semi-solid agarose supernatant and resuspend the pellets in
25 ml of 2* LB-glycerol (12.5%) (see Preparation of Media and
Reagents) per 250-ml centrifuge bottle. Remove 100 *l for estimation
of library titer and further characterization. Pipet the remainder into
1-ml aliquots. Store at -80°C.
10.
Perform 6 serial dilutions with 100 *l of the amplified library diluted
into 900 *l of LB medium. Plate 10 *l of the 10-5 and 10-6 dilutions
onto selection plates. Amplification of a primary library containing
1 * 106 total transformants should result in a stable library of at least
1 * 109 total transformants.
pCMV-Script® XR cDNA Library Construction Kit






--
Ben Holt
Assistant Professor
University of Oklahoma
Department of Botany and Microbiology
GLCH Rm 219
770 Van Vleet Oval
Norman, OK  73019
Phone (405)325-9018
FAX   (405)325-7619
http://www.ou.edu/cas/botany-micro/faculty/holt.html <http://www.ou.edu/cas/botany-micro/faculty/holt.html><http://www.ou.edu/cas/botany-micro/faculty/holt.html>



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