PCR Protocol

20-mer Polymerase Chain Reaction Procedure (for MJ Research Thermal Cycler)

Caution: When the main power to the Unit has been switched off, allow at least 5 sec before switching on again. Under no circumstance should the Unit be set to "To printer and Ram."

1. Turn on the PHC-2 and circulator at least 20 min before an experiment.
2. Position the sample PRT (Pretinum resistance thermometer) in one of the center holes in the block.
3. Press the <PAGE> key to move to Unit display Page 1.
4. Edit the program or pick up a program if the program has been set.
   Reaction time for Taq I Polymerase reaction of cotton probe DNA.
   Denature 93 C 30 sec
   Anneal 55 C 60 sec
   Extend 72 C 120 sec
   * Total cycle : 30
5. Heat a 0.7 ml MFT containing reaction mixture for 5 min at 95 C to denature the DNA completely.
   Reaction mixture
   10X Reaction buffer 5 �l
   dNTP 0.2 mM (4 �l)
   Primer 1 100 pM (2 �l) from 50nM,100 �l TE
   Primer 2 100 pM (2 �l) "
   Template DNA 250 ng
   H2O to a final volume 50 �l
6. Positioned the MFT containing DNA sample and add 2.5 unitTaq I polymerase (0.5 �l: 5 unit/�l; Perkin Elmer Cetus). To withdraw the required amount of Taq I polymerase from the stock, spin the stock tube 10 sec at 4 C just before or dilute the enzyme with H2O and withdraw the diluted enzyme.
7. Overlay the reaction mixture with 100 �l of light mineral oil.
8. Press the <RUN> key to start the program.
9. Withdraw the sample of the amplified DNA from the reaction mixture and analyze it by gel electrophoresis. If necessary, remove the oil by extraction with 150 �l chloroform. Transfer aqueous phase, forming a micelle near the meniscus, with a P-20 to a new MFT.

MJ Research thermal Cycler: 10-mer PCR for amplification of random genomic DNA fragments of Cotton

1. Turn on the thermor reactor (MJ Research, model PTC 100).
2. Edit (or choose a program if it has been set up) PCR Program.
   Tdenature = 95 C, 5 min; 1 cycle.
   Tdenature = 94 C, 30 sec,
   Tanneal= 38 C, 1 min, 45 cycle
   Textension= 70 C, 2 min;
   Textension=5 min; 1 cycle.
   T4C=4C;hold forever.
3. Prepare reaction mixture and template cotton DNA.

* Enzyme should be kept in ice or table-top freezer. 10X reaction buffer, dNTPand primers should be kept in ice after thawing. DNA in H2O is not sensitive as much as others. However, the DNA solution should not be kept at room temp for longer than 15 min.

1. Number on the top of 0.5ml-microfuge tubes (MFT).
2. Prepare reaction mixture (for example of 25 �l reaction mixture)
   1X conc.
   2.5 �l of 10X reaction buffer (Promega) -
   1.5 �l of 25 mM MgCl2 (Promega) 2 mM
   2 �l of 10 mM dNTP (Promega) 0.2 mM
   (or mixture of 0.5 �l of four 10 mM dATP, dCTP, dGTP, dTTP)
   2 �l of 2.5 mM random primer (Operon) 0.2 mM
   Template DNA 10-30 ng (20 ng is opt.)
   DNA+H2O 18 �l
3. After tapping or voltexing the reaction mixture with DNA in 0.5ml MFT briefly, spin (10 K, 5 sec).
4. Run the PCR program. While denaturing the DNA at 95 C for 5 min, prepare the Taq polymerase in H2O.

   For 25 �l reaction mixture in a 0.5 ml MFT, Taq polymerase 0.3 �l (1.5 Unit; 0.5 Unit/�l) dissolving in H2O 2.5 �l

5. While the PCR cycle is processing at Td=94 C, 30 sec (1st stage and 1st of 45 cycle), open the top of MFTs.
6. When the transition stage starts (between Td and Ta) of the 1st cycle, pause the cycle (push a decimal point button) and add a mixture of Taq polymerase (2.8 �l/tube) with EDP (25 �l liquid end) as quick as possible. Do not touch the top of reaction mixture in the tubes. Drip the Taq enzyme mixture at a distance of 1-2 mm from the mixture surface. If touched on the reaction mixture with MFTs, use a fresh tip.
7. Add 30 �l sterile mineral oil on the top of mixture in MFTs with EDP as quick as possible and unpause the cycle (push decimal point button). It takes about 5 hr 30 min to 5 hr 45 min to finish the whole cycle. If overnight is necessary after the cycle, set 4 C (time holding) after final Te.
8. After the cycle, add 2.5 �l of 10 X tracking dye, tap the tubes to mix the dye and bottom phase, spin (5K, 5 sec).
9. Loading the sample (10 �l/lane) on a gel (1.4 % agarous gel is usually good). Using a two- ladder gel (2 combs) is working good.
10. Run the gel (160 volt, 1 hr) until the 500 bp-dye reachs to the bottom of the gel.
11. Take a picture.

GeneAmp 9600(Perkin Elmer Cetus): 10-mer PCR for amplification of random genomic DNA fragments of Cotton

1. Turn on the thermal cycler (GeneAmp 9600).
2. Edit (or choose a program if it has been set up) PCR Program (Math 4).
   Tdenature = 95 C, 2 min; 1 cycle.
   Tdenature = 94 C, 30 sec,
   Tanneal= 38 C, 1 min, 45 cycle
   Textension= 70 C, 2 min;
   Textension=5 min; 1 cycle.
   T4C=4C;hold forever.
3. Prepare reaction mixture and template cotton DNA.

   * Enzyme should be kept in ice or table-top freezer. 10X reaction buffer, dNTPand primers should be kept in ice after thawing. DNA in H2O is not sensitive as much as others. However, the DNA solution should not be kept at room temp for longer than 15 min.
   1) Number on the top of 200 �l thin-wall tubes.
   2) Prepare reaction mixture (for example of 25 �l reaction mixture)
   1X conc.
   2.5 �l of 10X reaction buffer (PerkinElmer) -
   2 �l of 10 mM dNTP (PerkinElmer) 0.2 mM
   (or mixture of 0.5 �l of four 10 mM dATP, dCTP, dGTP, dTTP)
   2 �l of 2.5 mM random primer (Operon) 0.2 mM
   Template DNA 10-30 ng (20 ng is opt.)
   DNA+H2O 17 �l
   AmpliTaq 0.3 �l (1.5 Unit)
   (Voltex the AmpliTaq tube briefly before aliquoting the enzyme)

4. Secure the cap with the roller tightly.
5. Voltex the reaction mixture with DNA in the tube briefly. Spin the tubes with adaptors (10 K, 5 sec), if necessary.
6. Start the PCR program. It takes about 4 hr to 4 hr 10 min to finish the whole cycle. If overnight is necessary after the cycle, set 4 C (time holding) after final Te.
7. After the cycle, add 2.5 �l of 10 X tracking dye/25 �l reaction tube, voltex the tubes briefly to mix, spin (10K, 5 sec) if necessary.
8. Loading the sample (10 �l/lane) on a gel (1.4 % agarous gel is usually good). Using a two-ladder gel (2 combs) is working good.
9. Run the gel (160 volt, 1 hr) until the 500 bp-dye reachs to the bottom of the gel.
10. Take a picture.