Flint Group Protocols
(1) Clone Maxiprep.
Growing up colonies from a glycerol stock.
Day 1.
Inoculation of starter cultures using LB medium containing ampicillin.
If you are seting up 14 colonies to growing up. You must inoculate a starter
culture of 10ML LB medium containing Ampicillin (the selective antibiotic) with 5µl
of the glycerol stock of interest, i.e.
10ML LB
10μl Ampicillin
5μl glycerol stock
NB: The Ampicillin:LB ratio must always be 1:1000 with a final Ampicillin
concentration of around 50μg/ml. Therefore, when growing up 14 selected colonies,
you should prepare in a sterilised flask the following volumes:
140ML LB
140μl Ampicillin
After mixing, make 10ML aliquots using 14 different 50ML Falcon tubes – can
simply pour the LB/Ampicillin mix from beaker into each Falcon tube. Incubate at
37oC overnight in shaking incubator.
Day 2.
Dilution of the starter cultures into LB Amp media.
Must transfer 10ML cultures to the larger volume of 200ML. In our example,
of the 14 cultures grown overnight, only 7 were selected for further incubation in
200ML LB. Therefore, the following mix is prepared in a sterilised beaker:
7X 200ML (1400ML) LB
7X 200μl (1.4ML) Ampicillin
Measure out 200ML aliquots of this mix in 7 different glass flasks (can be 500ML or
1L in size) using a measuring cylinder.
Shake the contents of each 50ML Falcon tube to resuspend the bacterial culture and
pour into each flask. Cover with foil on top and incubate in the shaking incubator
overnight at 37oC.
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Flint Group Protocols
Day 3.
Harvesting the bacterial cells by centrifugation.
I. The bacterial cultures must be transferred to the plastic ‘buckets’ which fit
into the J2 centrifuge (10.5 rotor) after ~1 days’ incubation. Make sure the
buckets are balanced by weighing them – ensure all weights are the same
with the addition of distilled water. Furthermore, remember to label each
bucket with the appropriate clone name.
II. Using the 10.5 rotor, spin down the buckets at 3000RPM for 15 minutes in
the J2 centrifuge.
III. Pour off the supernatant into a container filled with Virkon. Be careful not to
disturb the pellet – make sure it is positioned at the bottom of the bucket
when pouring to minimise the risk of disturbing the pellet.
IV. These cell pellets can be frozen indefinitely until they need Maxiprepping.
Plasmid DNA Purification Using QIAGEN Plasmid Maxi
Kit.
You will find all the necessary reagents in the QIAGEN Plasmid Maxi Kit
found in the lab. You can prep a maximum of 6 bacterial pellets at any given time.
I. Put Buffer P1 and P3 on ice.
II. Resuspend each pellet in 10ML of Buffer P1 (containing RnaseA). Can use
a P5000 Gilson to completely disrupt the pellet, ensuring there are no
clumps remaining.
III. Add 10ML of Buffer P2 to each bucket, mix thoroughly by inversion 3 or 4
times and incubate at room temperature for 5 minutes (but no more than
this). The lysate should appear viscous after mixing.
IV. Add 10ML of chilled Buffer P3 to each bucket one at a time and mix
immediately by gentle inversion 4-6 times. A fluffy, white precipitate should
form and the lysate should appear less viscous. Place on ice and incubate for
20 minutes. Remember to switch on the J2 centrifuge at this stage and allow
it to cool to 4oC by making the appropriate setting and closing its lid.
V. Spin down the buckets in the J2 centrifuge at 10000RPM for 30 minutes at
4oC. Before loading the centrifuge, ensure that the samples are mixed again.
During this spinning step, Kimwipe tissues can be folded into quarters and
placed on the tops of 50ML Falcon tubes in such a way as to act as filters in
the next step of the protocol.
VI. Towards the end of the 30-minute spin, you can equilibrate the QIAGENtip 500 columns by pipetting 10ML of Buffer QBT into each one, allowing
the buffer to flow through and collect in the drip tray beneath (you must use
the blue QIAGEN column holder at this stage).
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Flint Group Protocols
VII. Once the 30-minute spin is finished, carefully pour the supernatant
containing DNA from each bucket through the Kimwipe tissues and into
labelled 50ML Falcon tubes. Any remaining white precipitate should be
removed in the Kimwipe tissues. Remember to add Virkon solution to the
empty buckets.
VIII. Apply the supernatant containing plasmid DNA to each QIAGEN-tip 500,
allowing it to flow through and collect in the drip tray. Any plasmid DNA in
the supernatant will be trapped in the columns. NB: The supernatants should
be loaded on to the QIAGEN-tips promptly.
IX. Wash each QIAGEN-tip 500 by adding 30ML of Buffer QC, allowing it to
flow through the columns and then pipetting another 10ML of the buffer into
the columns. This wash buffer removes unwanted contaminants from the
columns.
X. Once the wash buffer has completely run through the tips, the DNA must be
eluted from the columns using Buffer QF. Position a round-bottomed
polypropylene tube underneath each column to collect the solution
containing the DNA, making sure that both sides of each tube are labelled
with the name of the clone. Elute the DNA by pipetting 15ML of Buffer QF
into each column and wait until every last drop of the solution has dripped
into each tube. Remember to switch the J2 centrifuge back on, allowing it to
cool down to 4oC.
XI. Precipitate the DNA by adding 10.5ML of isopropanol (2-propanol) at
room temperature, mix and spin down using the J2 centrifuge (rotor 17.0) at
10000RPM for 30 minutes at 4oC.
XII. The pellet should be visible on the side of each round-bottomed tube, close
to its bottom. Mark the position of the pellet on each tube with a marker pen.
By pouring slowly, carefully decant the supernatant into a labelled 50ML
Falcon tube. It is wise to keep the supernatant in the meantime, at least until
the DNA has been recovered at the end of the procedure. If the pellet
becomes dislodged when decanting, then use a P1000 Gilson to carefully
pipette off the supernatant. It is often the case that no pellet can be seen – in
such circumstances, it is best to pour out the supernatant as slowly as
possible.
XIII. Wash the pellet in 5ML of room-temperature 70% ethanol, swirl around
the suspected pellet and centrifuge at 10000RPM for 10 minutes at 4oC
using the J2 centrifuge (rotor 17.0 again). Carefully decant the supernatants
as before, into the same 50ML Falcon tubes. Mark the new position(s) of the
pellet(s) in each tube, as they will probably have shifted their positions
during the spin.
XIV. Air dry the pellets for at least 5-10 minutes (they can be left to dry over
lunch, for instance), upside down on tissue to drain away any excess
isopropanol.
XV. Resuspend each pellet in a total of 400μl of MQ water, providing the pellet
appears substantial. To begin with, add 200μl of MQ and pipette up and
down several times in the marked area of the pellet. Transfer the
resuspended pellet to a 1.5ML Eppendorf tube, appropriately labelled with
the clone name, date and DNA concentration (obtained from the nanodrop).
Then add another 200μl of MQ to each round-bottomed tube and pipette up
and down several times over all the sides at the bottom of each tube.
Transfer the remaining MQ to each Eppendorf tube. Remember to wipe off
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Flint Group Protocols
the marker label on each round-bottomed tube with ethanol when finished.
Rinse the tubes in ddH2O overnight and autoclave in a suitable bag.
XVI. Nanodrop to quantitate the amount of DNA in each of the samples, before
freezing them. You are looking for a DNA concentration of ~1000ng/μl
for the subsequent transfection study.
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