Cleaver DNA Kit - Teachers Version (261443 bytes)

Cleaver DNA Kit - Teachers Version (261443 bytes)
Teachers Version
(with Gel Images)
Instructional DNA Electrophoresis Kit
CSLDNAKIT1
Version 03092015
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PACKING LIST:
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5 x DNA samples premixed with runSAFE DNA Stain (300μl of each) in
tubes labeled Sample A to E. Please place these in a -20C freezer for
long term storage.
Agarose powder
Electrophoresis buffer as a 10X concentrate
20µl Mini Pipette
Instructions
The packing lists should be referred to as soon as the kits are received to
ensure that all components have been included. The components should be
checked for damage when received.
Please contact your supplier if there are any problems or missing items.
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Gel Electrophoresis
Gel electrophoresis is a separation technique used daily in research laboratories
throughout the world. Using this method the presence and size of nucleic acids and
proteins in a sample can be determined.
Equipment required
Gel tank with trays, end dams and combs
(recommended for a class of 30 pupils TGT6XMINI six gel system. For smaller classes
use a four (TGT4XMINI) or one gel system (TGT1XMINI)
Electrophoresis power supply
(recommended for running at up to 150 volts TPS150V and for better separation of the
samples up to 250 Volts when using a nanoPAC300 and TGT6XMINI system only)
Blue light or UV transilluminator
(recommended SAFEVIEW-MINI)
Gel documentation
(recommended low cost image taking using GDH-BASIC and a mobile phone camera)
Adjustable micropipettes and tips (or fixed volume pipettes)
Balance
Measuring cylinder
Vortex mixer
Microwave or hotplate stirrer
Large beaker or jug
DNA samples
Microcentrifuge tubes
(please consult the Breckland Scientific catalogue for a full range of related laboratory
equipment)
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Step by Step Instructions:One hour before the class begins remove the frozen DNA samples labeled A
to E from the freezer and allow to defrost.
1. Make the buffer.
Buffer is supplied as a 10X concentrate. Dilute by first establishing how much
buffer is required for the tank volume and the gel volume by the particular gel
system you are using and adding 1 part 10X buffer to 9 parts water (distilled or deionised is best but tap water is fine). For our recommended system TGT6XMINI
dilute by measuring 200ml of buffer and adding to 1800ml of water. Stir.
2. Make the agarose gel.
Weigh out the required amount of agarose that will give a 1% gel for the gel volume
you will be using so for a 100ml gel weigh out 1g of agarose. For our recommended
TGT6XMINI weigh out 3g of agarose powder and mix with 300ml of diluted buffer
Heat in microwave for 3 minutes or until the solution starts to bubble, stir and then
follow by 30 second bursts until the agarose has completely dissolved. The solution
will be completely clear. Set aside to cool. Alternatively if you are dissolving the
agarose using a heater stirrer or water bath, this should be set to 65 degrees
centigrade and prepared beforehand.
3. Pour gels.
Different gel systems have different methods for casting gels including traditional
taping method, in tank casting and the use of casting systems. We recommend using
the simple leak proof tray and end dams system of the Cleaver TGT systems.
Instructions are below. For traditional tape casting please see page 8.
Cleaver TGT systems Leak proof end dam casting:Fit the end dams tightly to the gel trays. The best way of doing this is to place one
dam on the bench and then push down firmly with the tray. Then repeat for the
other end of the tray and dam. Insert the comb or combs depending on how many
samples you wish to load. We recommend that each pupil loads one sample so there
will need to be as many wells as required for the number of attendees. Each comb
contains 6 wells and each gel tray in the TGT systems has at least two comb slots .
Once the agarose has cooled to about 65ºc pour it into the gel trays so that the base
of the gel comb is submerged in the gel about 5mm – about 30 - 40ml of gel volume
per tray used. Leave the gels to set. As they set the gels will turn cloudy. This will
take about 20 minutes. Once set very carefully remove the comb(s) and end dams as
the gel is easy to tear.
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4. Prepare samples.
You have five DNA samples provided in micro-centrifuge tubes pre-mixed with
runSAFE stain DNA stain. These are labelled A to E. These contain marker dyes to
show how far the samples have migrated and actual DNA bands of different sizes
which will separate out during the run when the current is applied from the power
supply. Two of these samples are identical and the rest are different. The purpose of
this practical is to find out which two are identical. These should have been removed
from the freezer one hour before the class started. If not and these are still frozen
then gently warm. Vortex these samples for 5 seconds to ensure the DNA is mixed
with the dye and stain evenly and then micro centrifuge the tube for 5 seconds to
ensure the liquid is at the bottom of the tube. If you don’t have these equipments
then first shake the tube vigorously for 5 seconds then tap the tube on the bench for
5 seconds.
5. Load the gels.
Use a micro pipette to load 20 µl of the contents of DNA sample A into the first well,
DNA sample B into the second well etc, being careful to use a fresh tip each time so
as not to contaminate the samples. Image below shows a gel which has been dry
loaded. Please see suggested loading sequence.
Sample A
No Sample
Sample B
Sample C
Sample D
Sample E
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6. Run the gels.
The gels trays can now be carefully transferred to the electrophoresis tank. The
wells containing the samples need to be at the negative electrode (black electrode
cassette) end of the tank.
Carefully pour the diluted buffer into the tank reservoir being careful not to wash the
samples out from the wells.
Connect the leads by fitting the lid and set the power supply to 150v. Leave this to
run for about 25 minutes. This will give a good separation of the DNA bands.
However if you run the gel for 35mins or have a power supply with a higher
capability and have limited time we would recommend setting to 250 Volts to give
better separation of the sample bands. You can tell how far your sample has moved
by viewing the dyes that are loaded with the DNA.
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7. Visualise the gels.
You will see the electrophoresis dyes separate out (See Image above) on the gel but
these are just dyes and not the actual DNA. Stop the power supply, open the lid of
the tank. Carefully remove your gel from the tank keeping it in the transparent tray.
Blot dry or allow any excess buffer to drain off and then place on the Blue Light or
UV transilluminator. When using a UV transilluminator great care needs to be
taken to ensure that everyone is using the appropriate safety glasses or safety
face guards and exposure to the UV light is kept to a minimum. Viewing of the
gel is most convenient using the SAFEVIEW-MINI which has an orange filter in the lid
which enables the DNA bands to be seen. Lower the filter lid and the different sizes
of DNA can be seen as white bands stretching away from the wells. Photographs can
be taking with a camera or a mobile phone. Even greater visibility can be achieved
using GDH-BASIC basic gel documentation hood which has a slot for the camera.
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This can be placed over the top of the transilluminator and orange filter lid and then
looked through or use a mobile phone camera to take pictures.
Two of the lanes from A to E will have identical bands and therefore come from
the same sample.
Gel A
Gel B
Image A above is a 1% gel ran for 25mins at 150V. Image B is the same gel ran for
35mins at 150V. If you run the gels for longer you would see even better separation and
resolution.
Both Images are taken using an Iphone with the gel being viewing on the SAFEVIEWMINI
Using Traditional tape Gel Casting method:1. Autoclave or plastic backed general tape should be used. A length 5cm longer
than the width of each end of the tray should be cut. One length should be
placed over one end of the tray and stuck m1cm in from the tray edge. This
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should then be folded and the edges sealed securely. Repeat for the other end
and place onto a level surface for gel pouring.
2. Place the comb(s) in the grooves. Each tray has more than one comb grove so
that multiple combs can be used. Using multiple combs increases sample
number available per gel but decreases run length and care must be taken to
ensure that samples from the first wells do not migrate into the lanes of the
second comb wells.
3. Pour in the agarose carefully so as not to generate bubbles. Any bubbles that do
occur can be smoothed to the edge of the gel and dispersed using a pipette tip.
4.
Allow the agarose to set, ensuring that the gel remains undisturbed.
5. Carefully remove the gel casting gates and comb and transfer the gel including
tray to the main tank.
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Teaching Tips
It is more usual in laboratories to wet load the gels in situ in the tanks through the
buffer.
However when dry loading the samples students are more easily able to see the wells
and, if using a tank running multiple gels, it removes the bottleneck around the
tank. Where time is a factor, provide the students with ready prepared gels, these
can be made the day before and kept in the fridge. Buffer can be saved and used
more than once.
A black plate put beneath the gel tray can be used to enhance visibility of the comb
wells when loading the samples.
Additional safety controls and hints
Allow the agarose to cool before pouring as, if too hot, not only can it burn the skin if
spilt but the gel tray could become warped. Practice pipetting technique using coloured
water before pipetting the DNA samples.
Other available equipment:Gel tank with trays, end dams and combs and electrophoresis power supply
(BIO-070-100)
Blue light transilluminater (BIO-080-100)
Adjustable micropipette (eg PIT-200-200)
Tips (PIT-100-150)
Balance (eg BAR-050-150)
Measuring cylinder (CYP-050-002)
Vortex mixer (BIO-095-100)
Microwave or hotplate (HOT-700-100)
Large beaker or jug (MEA-150-002)
DNA samples (BIO-890-100)
Microcentrifuge tubes (CEN-050-150)
Buffer (BIO-870-036)
Agarose (BIO-350-050)
runSAFE stain (BIO-885-012)
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SAFETY PRECAUTION
WHEN USED CORRECTLY, ELECTROPHORESIS UNITS POSE NO HEALTH
RISK.
HOWEVER, ELECTROPHORESIS UNITS CAN DELIVER DANGEROUS LEVELS
OF ELECTRICITY AND ARE TO BE OPERATED ONLY BY QUALIFIED
PERSONNEL FOLLOWING THE GUIDELINES LAID OUT IN THE EQUIPMENT
INSTRUCTION MANUAL.
Version 2.9.15
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