Biology Reference
In-Depth Information
2.
Incubate. Separate the four plates to the four different temperatures being tested.
Allow the worms to develop into adults and lay progeny over a 24-h period.
3.
Transfer worms. Transfer each of the five worms to a new well in the same
12-well plate, 1 worm per well (labeled B1-B5). We leave the two center
wells empty to separate between A and B wells, and because these wells
tend to show different conditions from the outer wells.
4.
Incubate. Place the 12-well plates at their respective temperatures for another
24-h period. Then, remove the worms from the B wells.
5.
Score percent lethality. After well A or well B sits without adults for a 24-h
period, allowing time for any eggs to hatch, count the total brood as well as the
number of larvae and eggs for each well. Calculate percent lethality by dividing
the number of eggs by the total number of larvae and eggs and multiplying by 100.
Gene Target Selection
Depending on the project resources and the scale of the screen, the investigator
must determine which and how many RNAi clones to use, defining a ''target set.''
This set could encompass a library of genes selected by different criteria (i.e., GO
term category), or could be as large as all clones available to target the genome.
.uk/products/rnai/Celegans.jsp
) is the largest source of bacterial-feeding clones. The
collection comprises 16,757 clones divided into 52 384-well bacterial clone plates.
The ORFeome-RNAi v1.1 library developed in the Vidal Lab (
Rual et al., 2004
)
(
http://www.openbiosystems.com
) contains 11,511 RNAi clones divided into 1308
96-well plates. The latter collection includes clones for
1700 genes not currently
targeted by the Ahringer
'
s library. Combining both libraries, the overall predicted
coverage of the C. elegans genome is
88% of the gene models in WormBase
release WS200. Specialized feeding RNAi libraries are also available from
Geneservice (
http://www.geneservice.co.uk/
)
, which collects clones representing
genes for specific ''processes'' such as chromatin (257 clones), phosphatases (166
clones), and transcription factors (387 clones).
2. Preparing the Bacterial Library
To assemble a library of the target set from the bacterial-feeding collection, the
selected clones must be cherry-picked from their original plates and frozen onto new
plates. The plate and position of the clones can be obtained from WormBase (
http://
www.wormbase.org
)
, or from Geneservice (
http://www.geneservice.co.uk/products/
tools/Celegans_Finder.jsp
)
using the ''C. elegans finder'' tool for the Ahringer
library, or by searching a downloadable Excel file available from Thermo Fisher
Scientific Inc. for the ORFeome-RNAi v1.1 library (
http://www.openbiosystems
Search WWH ::
Custom Search