Xenopus Gene Collection (XGC) Plates
Please note that the plate(s) being sent have tested negative in
our phage contamination assay. However these plates should still be
handled with care as no phage assay can be guaranteed to be 100%
accurate
WHAT TO DO WITH YOUR CLONES WHEN YOU RECEIVE
THEM
IRBH (Xenopus laevis) and IRBN
(Xenopus tropicalis)
Plates have been replicated in LB broth containing 8% glycerol and
50µg/ml ampicillin.
The plates have been shipped frozen in dry ice and should be
placed in a -70°C freezer on arrival.
To make further copies replicate in the above media and incubate
overnight at 37°C.
IRBG (Xenopus
laevis)
Plates have been replicated in LB broth containing 8% glycerol and
27µg/ml chloramphenicol.
The plates have been shipped frozen in dry ice and should be
placed in a -70°C freezer on arrival.
To make further copies replicate in the above media and incubate
overnight at 30°C.
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Vector Information
In each of these subsets all clones on one plate share the same
vector. For details of vectors used follow these links:
IRBG (Xenopus
laevis)
IRBH (Xenopus
laevis
IRBN (Xenopus
tropicalis)
Vector information can be found here.
RECOMMENDED METHODS FOR ISOLATION AND PURIFICATION OF
DNA:
Plasmid DNA mini-prep
preparation
Any standard mini-prep method is
suitable. Below is the method used in-house at Source BioScience
LifeSciences.
a) Streak out plasmid onto LB + ampicillin (or chloramphenicol
[CM]) plate. Incubate overnight at 37°C (amp.) or 30°C (CM).
b) Dispense 5ml LB medium into a 50ml sterile tube. Add
appropriate antibiotic. Inoculate with a single colony and incubate
overnight in shaking incubator 37°C [30°C (CM)]/170 rpm.
c) [If a glycerol stock is required, transfer aliquot of 140µl to
a 1.5ml microtube. Add 40µl 80 % glycerol. Mix and freeze.]
d) Take 2ml of the plasmid broth, transfer to a 2ml microtube, and
centrifuge 15 min 3000 rpm.
e) Pour off supernatant and discard. Re-suspend pellet in 200µl
GTE. Add 5µl RNAase A stock (10mg/ml). Incubate 10 min. room
temperature.
f) Lysis: Add 400µl 0.2M NaOH/1% SDS (freshly made). Mix by
inversion. Place on ice for 5 min.
g) Add 300µl 3M K Ac. Invert to mix. Place on ice for 10 min
h) Centrifuge 10 min at 13000 rpm in microfuge. Decant supernatant
into a 1.5ml microtube
containing 1ml cold ethanol, vortex briefly and leave to stand for
30 min. on ice.
i) Centrifuge 10 min. at 13000 rpm in microfuge. Remove ethanol
carefully (preferably by suction technique).
j) Add 200µl cold 70% ethanol, vortex briefly, centrifuge 10 min.
at 13000 rpm in microfuge, remove ethanol carefully (as above).
k) Air dry pellet. Add 50µl TE and leave to re-suspend overnight
at 4°C.
Solutions:
10 ml GTE:
0.5 ml 20% glucose
0.25 ml 1M Tris/HCl pH 7.5
0.2 ml 0.5M EDTA pH 8.0.
9.05 ml sterile distilled water
Lysis Buffer:
100 ml 0.2M NaOH/1% SDS = 0.8 g NaOH
5ml 20% SDS
95ml sterile distilled water.
PEG purification of plasmid DNA (strongly recommended before
sequencing)
a) Add an equal volume of 20%
PEG solution in 2.5M NaCl. Incubate at room temperature for 5-10
min.
Spin for 10-15 min. at 13,000 rpm.
b) Remove supernatant and rinse pellet with 70% ethanol. Dry the
pellet. Resuspend in 20µl sterile
distilled water.
c) Remove 5µl and add to 2µl loading buffer. Run out with
appropriate DNA marker on a 1.5% TBE
agarose gel to check that DNA recovery is good.
d) The DNA is now ready for sequencing.
General PCR Purification Method
Clones can be amplified by PCR using
the appropriate flanking primers. PCR products should then be
purified using spin columns or the sAP/exo1 (shrimp alkaline
phosphatase/exonuclease1) method prior to sequencing.
Werle. E, Schneider. C, Renner. M,
Volker.M and Fiehn. W. (1994) Convenient single-step, one tube
purification of PCR products for direct sequencing. Nucl. Acids
Res. 22: 4354-4355.
Hanke. M and Wink. M. (1994) Direct
DNA sequencing of PCR-amplified vector inserts following enzymatic
degradation of primer and dNTPs. BioTechniques 17: 858-860.
Version 2.1 13th June 2005