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384-well PCR

Prepared by Anna Guerasimova

    Beforehand.

  1. choose empirically the annealing temperature of primers;
  2. make sure you have enough PCR-plates, Q-covers and sealing tape;
  3. prepare the stocks: PCR-buffer, 5M Betaine - with some excess, dNTPs, primers, Taq:Pfu mix (100:1 calculated by activity units) - with small excess;
  4. PCR-reaction.

  5. thaw the plates with bacterial culture;
  6. sign the 384-plates;
  7. prepare PCR-mix;
  8. dispense PCR mix by multichannel pipet or by "Multidrop 384" machine;
  9. centrifuge Q-covered (or sealed) plates at ~800 rpm, 3' NT;
  10. transfer the bacteria twice with the sterile replicators;
  11. seal the plate and run PCR reaction;
  12. After PCR.

  13. take the plates out with the help of screwdriver (or forceps);
  14. move the plates to +4oC and then to -20oC;
  15. centrifuge at ~800 rpm, 3' NT;
  16. control PCR by agarose gel electrophoresis: 0.5 - 1µl from 5 - 15 wells;
  17. seal the plates again;
  18. move the plates to +4oC and then to -20oC;
  19. for long term storage - -70oC (keep track in your lab book!).


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    Solutions.

    PCR buffer 10x

    (store at +4oC):

    Conc.Stock500ml
    Tris Cl, pH 8.6*0.5M1M250ml
    KCl0.5M2M125ml
    MgCl215mM1M7.5ml
    Tween 201%100%
    p=1.108
    5.0ml
    5.54g

    H2O mQ112.5ml
    Conc.Stock500ml
    Tris Cl, pH 8.6*0.5M1M250ml
    KCl0.5M2M125ml
    MgCl215mM1M7.5ml
    Tween 201%10%50ml
    H2O mQ67.5ml

    p (10x Buf.) = 1.018 g/ml
    * - another possibility: 175ml (1M Trise-base) + 75ml(1M Trise-HCl).

    PCR buffer 2.5x

    (store at +4oC)

    Conc.Stock100ml300ml
    Buffer (15mM MgCl2)2.5x10x25ml75ml
    Betaine3.75M5M74.75ml224.25ml
    Cresol red (Na)125 µM50mM250 µl0.75ml

    p (2.5x buffer) = 1.06g/ml

    PCR mix

    Store working quantity at +4oC; the main stock at -20oC. It is possible to keep PCR mixture with enzymes at at +4oC for about 15h without any decrease of efficiency of PCR reaction.

    Conc.Stock72.5ml**
    PCR buffer1x2.5x29ml
    dNTP’s0.2mM25mM580µl
    Primer#1 & #2200pmol/ml?? µl 
    H2O mQ?ml 
    Taq/Pfu pol***50-250u/ml?u/ µl?µl 

    ** - 72.5ml is enough for 9 plates [20µl in one well] (110ml for 30µl; 145ml for 40µl);
    *** - Taq(u):Pfu(u)=100:1.


    Comments.

    Detailed instructions, step by step

    #1.

  • As a first approximation for the annealing temperature of primers one can use the Tmelting value given by the program "Oligo". Take into account that betaine addition would probably decrease the annealing temperature (sucrose has no effect on it). For example, for the primer pair M-13/23 and M-13/30 maximum annealing temperature decreased from 71oC to 67oC when 1.5M betaine was added.
  • #3.

  • It is convenient to have:
    * all four dNTP's mix (100mM or 25mM stocks);
    * primers mix.
  • #4.

  • Frozen bacteria plates have to be placed on a bench in one layer one by one, separately. This prevents condensation of liquid on the inner side of the cover and transfer of liquid between wells. Condensation occurs when temperature of the cover becomes lower than temperature of the liquid in the plate. It is convenient to use an air fan to accelerate thawing.
  • #6.

  • 12.2ml per plate, if PCR volume is 30µl/well (maximum for old ABT-384 plates #TF-0384) or 16.3ml per plate, if PCR volume is 40µl/well (maximum for new ABT-384 plates #AB-0937).
  • Add the enzyme last. We were told that Pfu polymerase can destroy primers if PCR mix stored for too long at room temperature. Therefore, just before you add the enzyme, in front of you should be labeled PCR plates, the reservoir for multi-channel pipette, bacterial plates and the replicators. We have tested that it is possible to keep PCR mixture with enzymes at +4oC for about 15h without any decrease of efficiency of PCR reaction.
  • After the PCR mix is aliquoted into plates, transfer the remaining volume to 50ml Falcon tube and keep it at -20oC. You can use it for the subsequent experiments (with the same primers), thus minimizing the volume of new PCR master-mix which needs to be prepared.
  • #8.

  • Optional — remove air bubbles from bottom of the wells.
  • #9.

  • You need twice as much replicators as number of PCR-plates. Insert replicator from the nearest edge, holding it leaned at 5-10o so that the nearest pins are closer to the plate (the row "P" in the PCR plate). The pins should be directed to the center of the wells.
  • The purpose of double inoculation is not to increase amount of transferred material but rather to decrease the probability of no transfer at all.
  • It is better to mix bacteria in the plate by replicator before transfer. Try to avoid mixing in only one direction, because it can induce sticking of partually lysed bacteria to the needles of replicator.
  • The volume of media captured on one needle of the replicator is ca 0.2µl.
  • In theory, the plates with inoculated bacteria can be stored at -20oC before PCR up to a few weeks. However, do not freeze them on dry ice. Instead, just put them in the freezer or on the towel soaked with the liquid nitrogen. If freezed on dry ice, evaporating CO2 will shift the pH. If betain- or sucrose-containing PCR mix was used, most likely PCR would succeed anyway, but you don't want be unsure about it, do you?
  • The same precautions should be taken for the PCR/NaOH mixtures for spotting. It is possible to freeze them in dry ice (for about 0.5 - 1h) only if they are sealed by "Tesafilm" or "Adhesive Sealing Sheet".
  • #10.

  • Sealing films: the best is the office tape from TESA (Tesafilm #2826775): (i) easily taped and removable; (ii) reliable; (iii) cheap - about 10DM per roll. Much worse variants:
    • "Adhesive Sealing Sheet" from ABgene: (hardly removable, expensive);
    • Microseal "A" film. Fragile: don't press the PCR machine cover very tightly, it can damage Microseal "A", it shrinks after PCR and unsuitable for freezing. For Microseal "A" film:
      • when PCR is finished - immediately (plate still in the PCR block) take off the upper film;
      • take the plates out of PCR machine;
      • remove the isolating film layer;
      • get rid of the PCR mix which is in between wells with the yellow tip connected to the vacuum pump;
      • use soft blotting paper to remove liquid from the plate surface;
      • cover with the "Genetix" covers;
    • "Bio-stat" film. It has to be sealed tightly, e.g. using the backside of the marker to help. One can put a piece of thin rubber on top, as well. Funny: there were cases when PCR's worked fine with a quite sloppily attached Bio-stat film. In that case considerable volume was evaporated uniformly from each well. Probably there is some sort of equilibrium inside the PCR block so that the content of the well does not dry out completely.
  • For 384-PCR it is better to change the parameter exceed of temperature of the lid "Setup/Lid/Tracking/Lid offset" from the default 15oC to 5oC.
  • PCR-mix: our impression, that in general betaine is slightly worsening PCR reaction. The enzyme speed and activity became lower. But it is impossible to exclude betaine from the buffer for 384 plates. Without it we observed smear, differing from PCR plate to plate (in an 8-strip from the same PCR-mix PCR worked fine). Adding of sucrose has similar effect - the smear partially disappeared, but with betaine - completely.
  • If a sub-cycle PCR is used (see below) the product yield is more stable and a little bit higher (probably it is because of low-melting regions in some sequences). We used the following program:
    Step:T (oC):Time:
    1951:00
    2940:15
    3590:15
    4650:55
    5go to 32 add. times
    6go to 229 add. times
    76810:00
    840
    9END 
  • #11.

  • Lift each four edges carefully and hold the plate with your hand. Avoid the abrupt moves, if not, your PCR ends up on the cover.
  • The plate holder for 384-plates can be treated with "Teflon spray", then the plate can be removed easier.
  • #14.

  • Concentration should be more then 0.15µg/µl.


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Last modification: 30/07/13

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