Home

Description

Search Catalogues

Browse catalogues

Collections

Prices

Guidelines
CELL LINES
PLANT CELL LINES
PLANT VIRUSES
PLASMIDS
PHAGES
MICROORGANISMS
GENOMIC LIBRARIES
PROCEDURES
CATALOGUE

Search Web Site

Contacts

FAQ

NB: These are examples of a PCR (= Polymerase Chain Reaction) ONLY. If a different set of primers are used there are many things that may have to be changed:

1. The correct primer concentrations required. (this will have to be determined by using various concentrations on total human genomic DNA - thus determining the optimal concentration)
2. The correct final concentration of magnesium (Mg²⁺ ) required in the PCR mix.
3. The temperatures required for the denaturation, annealing and synthesis steps to be programmed into the PCR machine.
4. Whether the PCR requires a 'hot start' (the TAQ polymerase is added after the initial denaturation step) or a 'cold start' when the TAQ polymerase can be added together with the master mix at the start.
5. The % agarose concentration needed for the gel stage to 'see' the PCR products will depend on the size of the amplified product ( the amplified product is the newly synthesised DNA that has been produced by the PCR reaction).
6. The molecular weight DNA markers will have to be selected as appropriate for the size of the amplified product.

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

The reason that we need to test out a new batch of DNA agarose pellets by PCR is because that is exactly what the users will be doing. So after making new pellets, they should be tested, especially if the cell pools which are being used to make them are old.

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

EXAMPLE 1

Materials Required

50ml Falcon tubes to wash the 'pellets' before the PCR
1x TE (see SOP GLS/07)
Agarose 'pellets': 1^o pool 11(old 'plugs'), 1^o pool 11 (new 'pellets') and 2^o pool 'pellets' 11/7
Waterbath set at 65^oC
Ice in ice bucket
Perkin Elmer PCR tubes, lids and frame
Sterile microtubes
Sterile pipette tips
Eppendorf pipettes
"Roller" to seal PCR lids on tubes

Positive control - human genomic DNA [kept at -20^oC in library PCR box]
Negative control - yeast DNA [kept at -20^oC in library PCR box]

Perkin Elmer Cetus - #YAC 2 programme 79
2% agarose (electrophoresis grade: Gibco #15510-019)
Microwave oven (Toshiba, ER-8730)
1x TBE (diluted 1 part to nine parts milliQ water from 10x stock TBE [National Diagnostics #EC-860])
Ethidium bromide 1mg/ml stock
Gel loading buffer = 15% Ficoll in 1x TE + bromophenol blue
DNA marker VI
Biorad mini sub DNA cell
Gene power supply GPS 200/400
UV illuminator and photographic system

Method

WEAR GLOVES THROUGHOUT

Washing the agarose pellets

The pellets have to be washed, to remove any inhibitory products that may have accumulated in them. We use Dr May Tassabehjis' protocol:

1. Place each pellet in a separate, labelled, sterile 50ml Falcon tube. Add 30ml of 1xTE.
2. Shake at RT, changing the buffer twice, allowing at least one hour per wash
3. Leave overnight at 4^oC in fresh TE

Melting the agarose pellets

1. Place the washed pellets in labelled, sterile 1.5ml microtubes
2. Place in a polystyrene float, and melt by placing in a 65^oC waterbath for 10-15 min.

Setting up the PCR reaction

1. Collect an ice bucket with ice
2. Thaw the required reagents (but not the TAQ) from the PCR box in the -20^oC freezer: the dNTPs, Mg²⁺ buffer, human genomic DNA and yeast DNA. Thaw as rapidly as possible, and then place on the ice.
3. Make the primer dilutions: 1 in 20 in autoclaved milliQ water.
   e.g. 10µl primer in 190µl water, in sterile labelled microtubes.
   Place on ice

Making a master mix

Setting up your samples

The PCR machine

PCR Cetus YAC2 program 79

1. Switch on the PCR machine [called YAC2] half an hour before you are ready to use it
2. Take your ice bucket with the PCR tubes and the TAQ to the PCR machine. You will also need to take a second strip of caps, and a 0.05ml combitip with a Gilson microtip at the end and the dispenser set at 5 (this will dispense 5µl)
3. Start the PCR machine by selecting RUN, <enter>, set the volume to 25µl <enter> and after the message close the lid and tighten, the PCR program will start.
4. When the temperature has reached 80^oC, open the lid, remove your PCR tray from its brown base, sit on the machine, and close and re-tighten the lid. The program will reach 94^oC, and count down from 5 minutes.
5. When only 10 seconds remain, press PAUSE.
   Now you can open the lid and remove the lids from the PCR tubes.
   Fill the combitip with the TAQ dilution, and add 5µl to each tube. Make sure each tube is well mixed. Replace with the new caps, and use the cap sealer to tighten them on
6. Close and re-tighten the lid, and press RUN to restart the program.
7. At the end of the PCR run, remove the plate and switch off the machine.

Running the agarose gel

In order to see the PCR products they are run (electrophoresed) on an agarose gel, the DNA stained with ethidium bromide, and visualised under UV light.

1. After ensuring that it is clean, tape up the open ends of the smallest size gel tray with autoclave tape. It is easier to remove the tape afterwards if the ends are turned over.
2. Place the gel tray on a level bench. and place the well comb, with 13 spaces, above the gel tray, so that the wells will be about 1/4 down from the top of the gel.
3. Weigh out 1g of the agarose, and place in a 100ml Duran bottle with 50ml of 1 x TBE buffer. Mix. Microwave in several batches of 1 min, setting 3, until the agarose is fully dissolved.
4. Allow to cool slightly, and then add 25µl of ethidium bromide
5. Pour into the gel tray and allow to set
6. When set, remove the tape from the ends of the gel, and place the gel on its base into the gel apparatus. Remove the comb gently. The red (+ve) electrode should be at the end closest to you, with the wells towards the top of the gel. Pour over 1 x TBE until the gel is completely covered. Add 150µl of ethidium bromide to the buffer and mix into the buffer using a plastic pasteur.
7. Add 7µl of the gel loading buffer to each of the PCR tubes.
8. Leaving the first well free, load 12µl of the PCR mixture into each well. Use the pipette to mix in the loading buffer before removing each 12µl. Continue until all 10 of your PCR reactions are loaded.
9. To well 1 and 12, add 5µl of DNA marker VI.
10. Place the lid on, and connect the electrodes to the power supply. Make sure that the red lead is always connected to the red terminal, and the black to black.
11. Switch the power supply on at the back, and also at the front. Adjust the voltage so that it reads 80 volts.
12. After a few minutes check that the samples are moving in the right direction i.e. towards you (= into the centre of the gel).
13. Allow the gel to run for 1.5 to 2 hours (but not so long that the front runs off the gel)
14. When finished, switch off the power before attempting to remove the gel.

Carefully remove the gel from the electrophoresis tank, lay it on a plastic sandwich box lid, and carry it to the darkroom.

1. Switch on the Viglen monitor
2. Switch on the Mitsubishi monitor
3. Switch on the video copy processor (the power switch)
4. Switch on the UV transilluminator
5. Place the gel directly onto the transilluminator (wear gloves) directly under the camera (the gel must be removed from the plastic gel forming tray)
6. Click the mouse to get the image in the screen
7. Adjust the camera height to get the full gel image on the screen
8. Adjust the focus/brightness on the camera to get a sharp bright image
9. Click the mouse to set the image and remove the lines
10. Press print to get the photograph
11. Clean up all the buffer from the transilluminator after use

EXAMPLE 2

Proceed exactly as in the above example except that:

PCR Cetus YAC2 program 82