Our cDNA libraries were constructed
in pBluescript SK- (Stratagene) using the Uni-Zap XR Vector kit. Phagemid
libraries were converted to plasmid libraries via mass excision, using
XLOLR as the plating strain of E. coli. Colonies were picked robotically
with a Q-bot robot (Genetix), and cultivated in 384 well microtitre plates
(see freezer medium).
In our project, clones are selected for
expression analysis based on sequencing results. Selected clones are rearrayed
in 96 well plates containing
freezer medium
to make glycerol stocks for the microarrays. To grow bacteria for production
of DNA for the arrays, cultures are grown overnight in LB medium in a 96
well format, and the clone inserts are amplified from the bacterial cultures
with out purification of the DNA. The PCR amplification conditions are
given below.
| Components | Volume | Final concentration |
|---|---|---|
| 10 x buffer* | 5 ul | 1x |
| MgCl2 (25mM) | 3 ul | 1.5 mM |
| dNTP (10 mM) | 1 ul | 200 mM |
| Primers**: | ||
| T7 22-mer primer OD: 1.0 | 1 ul | |
| SK 20-mer primer OD: 1.0 | 1 ul | |
| Taq polymerase | 0.2 ul | 1 unit |
| sterile water | 36.8 ul | |
| template bacterial culture | 2 ul | |
| Total | 50 ul |
* We use buffer from Promega.
** T7 and SK are sequencing primers, specified
by Stratagene, that flank the insert. We prefer SK over T3 because it is
closer to the cloning site. We make stocks of each primer with O.D. = 1.
T7 22-mer primer = 5' GTAATACGACTCACTATAGGGC
3'.
SK 20-mer primer = 5' CGCTCTAGAACTAGTGGATC
3'
| First denaturation: | 95oC for 10 minutes |
| 35 cycles of: | 94oC
for 30 seconds
60oC for 1 minute 72oC for 1 minute |
| Final Polymerization: | 72oC for 4 minutes |
| Hold at: | 4oC |
Check success of amplification by running 5 microliters (5 ul) of amplification products on an agarose gel.
2000.05.20
Aeroponic
chamber|Cultivation|DNA
extraction|EMS Mutagenesis|
Freezer
Medium|PCR Amplification|Seed
Germination|Seed Mill|
Medicago.org