Project Documentation & Protocols: Maize Gene Discovery Project: RescueMu Library Plates: Screening
Contents: Index | Progress | Ordering | Screening | Sequences | Phenotypes | Protocols | FAQs
Upon receipt of the 96-well library plate and prior to opening of the cover, please make sure the content is on the bottom of each well by either centrifugation or firmly tapping several times on a solid surface. Storage of small aliquots in individual microcentrifuge tubes is recommended to avoid repeated freezing and thawing.
PCR Screening of a Library Plate:
The primary use of library plates is to screen by PCR to search for RescueMu insertion events in defined targets. To do this you will need a Mu1TIR readout primer and a primer in a favorite gene or motif. You can multiplex gene primers to search to the right and to the left of potential insertion sites and you can mix primers from different genes together. We recommend running a control reaction in advance with complex primer sets to determine that you do not generate product in the absence of template. Germinal events can be identified by finding hits in both rows and columns. Seeds from corresponding individual plants are available at the Maize Co-op.
Mu1 readout primer for RescueMu
"RIGHT" 5' TAT TTC GTC GAA TCC GCT TCT 3'
"LEFT" 5' CAT TTC GTC GAA TCC CCT TCC 3'
Favorite gene primers: your own design
Positive Control gene primer: parental insertion launching sites (grid-specific)
||Recommended control primers
||Parental #1 5' TTT GTT GGT TCT GGG TTA AAG 3' plus
RescueMu RIGHT expect an 0.5 kb fragment
Parental #2 5' AAA TGG CAT TGA AAT CTA CTT 3' plus
RescueMu LEFT expect an 0.6 kb fragment
Recommended PCR Conditions
0.5 mM dNTPís
2.5 mM Mg2+
0.8 µM gene primer
4.0 µM RescueMu primer
2 units Taq polymerase
5 ng library plate DNA
||Number of cycles
|95oC, 5 minutes
|95oC, 30 sec/ 55oC, 30 sec/ 72oC, 2 min
|72oC, 5 minutes
|4oC holding until analysis
Note: for your "favorite gene," additional optimizations may be required.
Other Screening Possibilites
Each well of a library plate contains a few hundred to a few thousand RescueMu plasmids. An entire plate may contain up to 250,000 plasmids. If you want to recover individual plasmids, you may want to make a "bacterial library plate" by transforming into E. coli (we recommend using a bacterial strain suitable for eukaryotic genomic sequences, e.g., DH10B. This bacterial library can be screened by plating and colony lift hybridization to identify plasmid of interest. For example, a partial EST sequence of a gene can be used as a probe to find a plasmid containing the entire gene, its promoter, and 3' downstream region. Given the large size of rescued plasmids, many entire genes are represented in the libraries.
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