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LABORATORY PROCEDURES FOR GENOMIC LIBRARIES

REFERENCE NO: GLI/1998/3/12


TITLE: QUALITY CONTROL: PCR ON YAC POOLS



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

PCR example:

Positive clone

ICI

11B-G10

 

Primer pairs

5071

agc aac aca tat cag ggg c

 

 

5072

tgt agg ttg acc tta agg c

 

Product size

350bp

(base pairs)


Materials Required


50ml Falcon tubes to wash the 'pellets' before the PCR
1x TE (see SOP GLS/07)
Agarose 'pellets': 1o pool 11(old 'plugs'), 1o pool 11 (new 'pellets') and 2o pool 'pellets' 11/7
Waterbath set at 65oC
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

PCR reagents:

2mM dNTPs

[kept at -20oC in library PCR box]

 

15mM Mg buffer

[ " " ]

 

primer 5071

[ stock @ 0.51mg/ml kept in 4oC fridge]

 

primer 5072

[ stock @ 0.59mg/ml kept in 4oC fridge]

 

autoclaved MilliQ water

 


Positive control - human genomic DNA [kept at -20oC in library PCR box]
Negative control - yeast DNA [kept at -20oC 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 4oC 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 65oC 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 -20oC 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. 10Ál primer in 190Ál water, in sterile labelled microtubes.
    Place on ice


Making a master mix

Individual PCR reaction volume


12x master mix volumes (always make more than the number of samples)

 

 

2.5Ál 2mM dNTPs

30Ál

2.5Ál 15mM Mg buffer

30Ál

1Ál primer 5071 1/20

12Ál

1Ál primer 5072 1/20

12Ál

8Ál sterile MilliQ water

96Ál

  1. Label a sterile microtube with master mix, and carefully add the correct volumes of the reagents, using sterile tips, and keeping the tube on ice
  2. The dNTPs and Mg buffer stock tubes can now be re-frozen, and the primer stocks returned to the 4oC fridge.


Setting up your samples

Number

sample

Ál sterile MilliQ

Ál of sample

 

 

 

 

1

blank (water)

5

0

2

human genomic DNA

0

5

3

yeast genomic DNA

0

5

4

old pellet 11

4

1

5

old pellet 11

3

2

6

old pellet 11

1

4

7

new pellet 11

4

1

8

new pellet 11

3

2

9

new pellet 11

1

4

10

SRxSC pellet 11/7

3

2

  1. Load the Perkin Elmer rack with the PCR tubes, clipped in with the retaining cover on the top. Place on the ice.
  2. Add the MilliQ water to the appropriate tubes.
  3. Take each melted agarose pellet out of the water bath and add the appropriate amounts to the correct tubes.
  4. Add the human genomic DNA and the yeast DNA to the right tubes.
  5. Each tube will now have 5Ál of sample. Add 15ml of the master mix to each tube.
  6. Put the strip of caps on top of the row of tubes, press them down, and then roll over the caps with the special cap sealer to make sure each fits tightly. (If the tops are not well fitted, the high temperatures of the thermal cycler[the PCR machine] will evaporate the samples). It is easier to fit the caps tightly on with the PCR rack on the bench, but return it to the ice as soon as this is completed.
  7. As this PCR involves a 'hot start' the TAQ dilution can now be made.
    Each tube will need 5Ál of TAQ dilution to make the final required tube volume of 25Ál.
    Each tube will need 0.2Ál of TAQ
    Remove the TAQ from the -20oC. It will not be a frozen solid, as the enzyme is stored in glycerol. However there is such a small volume in the TAQ tube, and it is so expensive, that it is valuable to give the tube a short pulse spin in the microcentrifuge to ensure that all of the enzyme is at the bottom of the tube.
    Add 2.4Ál TAQ to 57.6Ál sterile MilliQ water, on ice
    Mix well, so that the thick glycerol is freely mixed with the water
    Keep the tube on ice so that it is available to add to the tubes after the initial denaturation step of the PCR cycle has been completed.
    The essential point is that the TAQ should be out of the freezer for the minimum time possible. Hence, refreeze as soon as the dilution has been made.


The PCR machine

The PCR program required is

94oC 5 min

30 cycles of

94oC 1 min/52oC 1 min/72oC 1 min

 

72oC 10 min

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 5Ál)
  3. Start the PCR machine by selecting RUN, <enter>, set the volume to 25Ál <enter> and after the message close the lid and tighten, the PCR program will start.
  4. When the temperature has reached 80oC, 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 94oC, 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 5Ál 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 25Ál 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 150Ál of ethidium bromide to the buffer and mix into the buffer using a plastic pasteur.
  7. Add 7Ál of the gel loading buffer to each of the PCR tubes.
  8. Leaving the first well free, load 12Ál of the PCR mixture into each well. Use the pipette to mix in the loading buffer before removing each 12Ál. Continue until all 10 of your PCR reactions are loaded.
  9. To well 1 and 12, add 5Ál 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.

NB: Everything that has come into contact with ethidium bromide must be disposed of very carefully, as this is a potent mutagen. Solids (e.g. tips) should be placed in a sweetie jar for subsequent incineration, whilst liquids should be carefully poured into the labelled Winchester for safe disposal.

Photographing the gel

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

NB: Wear the protective visor whilst in the dark room, working with the UV transilluminator. UV is very damaging to the eyes, so your eyes must be protected at all times.

  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

PCR example:

Positive clone

ICI

11D-A12

 

Primer pairs

6175

ctt tga aga tgc tcc caa tg

 

 

6176

tca ctc ata gga ggg caa ac

 

Product size

164bp

(base pairs)

Proceed exactly as in the above example except that:

1. The PCR program required is

94oC 5 min

30 cycles of

94oC 1 min/56oC 1 min/74oC 1 min

 

74oC 10 min

PCR Cetus YAC2 program 82

2. As the product is smaller than in example 1


- make a 2.5% agarose gel
- use DNA marker set V


Guidelines prepared for CABRI by HGMP, December 1998
Page layout by CERDIC
Copyright CABRI, 1998

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