What to do with your clones when you receive
them
Please note that the clone(s) being sent have tested
negative in our phage contamination assay. However, these clones
should still be handled with care as no phage assay can be
guaranteed to be 100% accurate.
Clones with the prefix LLAM
[example 12226314
(LLAM3083-n11)]
The clones have been streaked onto LB agar containing 50 µg/ml
ampicillin.
Please store them at 4°C (not in a freezer).
As soon as possible, restreak the clones onto the same medium
and incubate overnight at 37°C to obtain single colonies.
Clones with the prefix
LLCM [example 2841899
(LLCM16-b12)]
The clones have been streaked onto LB agar containing 20 µg/ml
chloramphenicol.
Please store them at 4°C (not in a freezer).
As soon as possible, restreak the clones onto the same medium
and incubate overnight at 37°C to obtain single colonies.
Clones with the prefix
LLKM [example 4233943
(LLKM1-d8)]
The clones have been streaked onto LB agar containing 30 µg/ml
kanamycin.
Please store them at 4°C (not in a freezer).
As soon as possible, restreak the clones onto the same medium
and incubate overnight at 37°C to obtain single colonies.
Clones with the prefix IR [example 4824526
(IRAT25-c6)]
These clones are the re-arrayed libraries and use
different antibiotics.
The clones have been streaked into LB agar containing their
respective antibiotic.
Please store them at 4°C (not in a
freezer).
As soon as possible, restreak the clones onto the same medium
and incubate overnight at 37°C to obtain single colonies
******************************************
A selection of at least 10 single colonies should be picked and
grown in LB broth containing the appropriate antibiotic + 8 %
glycerol for subsequent freezing at -70°C.
These frozen stocks can then be regrown for plasmid isolation and
purification.
Streaking out for single colonies is essential in case of
contamination or cross-contamination, or if modifications of the
original clone have taken place as the library has been replicated.
If there is a problem, the more single colonies you streak out, the
more likely it is that you can retrieve the original construct. You
can also isolate the original clone from a contaminated sample by
following this procedure.
WHAT TO DO IF YOU DON'T THINK YOU HAVE THE CORRECT
CLONE:
We have received reports that a small number of I.M.A.G.E.
clones distributed by the Resource Centre did not contain the
expected sequence. Please contact us if the clone you have received
is not what you expected. We are happy to supply the clone again,
in case there has been a mistake at our end.
However, before requesting a new clone, please read the
instructions on our WWW pages carefully to ensure that you used the
correct I.M.A.G.E. ID when ordering. Many cases of "wrong sequence"
have been caused by using an incorrect ID when the clone was first
requested.
Unfortunately, if the clone is still incorrect, this is beyond
our control, as the libraries were constructed and replicated
elsewhere.
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.
- Streak out plasmid onto LB + ampicillin (or chloramphenicol)
plate. Incubate overnight at 37ºC (amp.) or 30ºC (CM).
- Dispense 5ml LB medium into a 50ml sterile tube. Add
appropriate antibiotic. Inoculate with a single colony and incubate
overnight in shaking incubator 37º C [30ºC (CM)] /170 rpm.
- [If a glycerol stock is required, transfer aliquot of 140µl to
a 1.5ml microtube. Add 40µl 80 % glycerol. Mix and freeze.]
- Take 2ml of the plasmid broth, transfer to a 2ml microtube, and
centrifuge 15 min 3000 rpm.
- Pour off supernatant and discard. Re-suspend pellet in 200µl
GTE.
- Add 5µl RNAase A stock (10mg/ml). Incubate 10 min room
temperature.
- Lysis: Add 400µl 0.2 M NaOH/1% SDS (freshly made). Mix by
inversion. Place on ice for 5 min.
- Add 300µl 3M K Ac. Invert to mix. Place on ice for 10 min
- Centrifuge 10 min at 13000 rpm in microfuge. Decant supernatant
into a 1.5ml microtube containing 1 ml cold ethanol, vortex briefly
and leave to stand for 30 min on ice.
- Centrifuge 10 min at 13000 rpm in microfuge. Remove ethanol
carefully (preferably by suction technique).
- Add 200µl cold 70% ethanol, vortex briefly, centrifuge 10 min
at 13000 rpm in microfuge, remove ethanol carefully (as
above).
- 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 20 µl sterile distilled water.
c) Remove 5 µl and add to 2 µl 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.
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 1.3 23rd January
2001