CSS451 Spring 2010 Polymerase Chain Reaction Laboratory The purpose of the polymerase chain reaction (PCR) is to amplify specific segments of DNA. If one knows the DNA sequence of regions of DNA that flank a DNA segment of interest, it can be amplified. In this lab we will exploit the PCR process to amplify a segment of the cry1ia1 gene in putative transgenic plants. The use of PCR to detect transgenes in our plants is an efficient screen because only small amounts of DNA need be isolated (DNA quickprep), then the thermocycling is performed (3 hours), and then the sample is electrophoresed. This whole process can be completed in 1 workday for up to 96 samples. To conduct this PCR, we have synthesized flanking primers for the cry1ia1 gene. These primers will be combined with the template DNA, Taq polymerase, buffers and nucleotides and placed in a thermocycler for 30 cycles of amplification. If a 1.1 kb fragment is amplified, we can assume (with some level of confidence) that the gene is inserted into the plant genome. We visualize the amplified fragment via DNA electrophoresis. As part of the laboratory exercise, we will set up PCR amplifications to test putative transgenic potato lines and the progeny of a cross with our putative transgenic line. (A positive PCR provides strong evidence that the plant is transgenic, while no amplification indicates it is nontransgenic.)
Amplification of Genomic DNA using The Polymerase Chain Reaction DNA DILUTION For NPTII amplification, your DNA concentration should be no more than 100ng/ul, however, it is very important that the EDTA concentration be no more than 0.1 um in the final dilution for PCR. (See note at end of PCR protocol on a method to dilute your DNA if you need assistance or ask the TA.) PCR AMPLIFICATION (general background) Critical advice! Remember to keep the enzyme on ice at all times except when pipetting it into the master mix. Also it is imperative to maintain clean, sterile conditions when setting up the PCRs. Remember to wear gloves. 1. Thaw the buffer, dntp solutions and primers, and place on ice. Centrifuge briefly. Count the number of tubes you will use, adding one to this number to account for pipetting error. Aliquot the appropriate amount of each of the components into a 1.5ml eppendorf tube in this order: ddh 2 O, Amplitaq buffer, MgCl 2, dntps, and primer 1, primer 2, Amplitaq enzyme. The amount of each component used per reaction tube is given below: ddh 2 O 14.5 x ul 10x buffer 2 ul 50 mm MgCl 2 (if necessary) x ul 10 mm dntp mix 1 ul 20 um Primer 1 0.5ul 20 um Primer 2 0.5 ul (5u/ul taq polymerase) 0.5ul The final reaction volume is 20ul. 2. Aliquot the master mix (99ul/ tube) into 0.5-ml thin-walled 8-strip tubes and add 1ul (100ng) of template DNA(s) to the side of the tubes. Make sure that all the solution is at the bottom of the tubes before proceeding with the next step. 3. Turn on the thermalcycler 10 to 15 minutes before using the machine. Program the proper files for NPTII amplification. The NPTII amplification program consists of 1 cycle at 94ºC for 4 minutes, then 35 cycles at 94ºC for 30 seconds, 60ºC for 30 seconds, then 72ºC for 30 seconds. The machine will then hold the tubes at 4 o C or remove the tubes from the thermalcycler after the program is finished and store at 4C.
NOTE: Procedure to dilute stock DNA solutions when in T 10 E 1 1. Label your tubes. Estimate how many microliters of DNA you need in order to have 10ug total per ml of T 10 E 0.1. This is easily done by dividing 10,000 by the flourometer reading to determine how many ul of the concentrated DNA to add to the T 10 E 0.1. 2. Add 100ul of T 10 E 1 to a 1.5ml eppendorf tube, and then add (900ul-DNA volume) of 10mM Tris HCl. 3. Add the proper amount of DNA to the tube. Refrigerate. QUESTIONS TO THINK ABOUT: 1. What is the purpose of? dntps template DNA primer Taq polymerase 2. Why are two primers for the NPTII amplification? 3. Why is it very important to maintain sterile conditions in setting up PCR reactions? 4. Why are we concerned with the EDTA concentration in the amplification reaction?
Lab exercise PCR Protocol for the cry1ia1 Gene in Potato Component 2X Promega PCR Master Mix Amount per Reaction 12.5 µl Cry1Ia1-F (forward primer) (20mM) 1.0 µl Cry1Ia1-R (reverse primer) (20mM) 1.0 µl BCH-F (forward primer) (20mM) 0.3 µl BCH-R (reverse primer) (20mM) 0.3 µl nuclease free water 7.9 µl 23.0 µl *Use 23 µl of the above mixture plus 2 µl of DNA for a total reaction volume of 25 µl *The ideal annealing temperature for the cry1ia1 and BCH primers is 50 C so use program KZ50 on the thermal cycler Step 1: 94 C: 5 minutes Step 2: 94 C: 1 minute Step 3: 50 C: 1 minute Step 4: 72 C: 1 minute, 30 seconds Step 5: Repeat steps 2 through 4, 30x for a total of 31 cycles Step 6: 72 C: 10 minutes Step 7: Hold at 4 C Fig.1 Population segregating for the cry1ia1 gene (top band); lane2: positive control, lane 3: negative control *The cry1ia1 band should be 1.1 kb and the BCH internal control band should be 290 bp (the DNA ladder at the left is a 100 bp ladder)
PCR Protocol for the nptii Gene in Potato Component 2X Promega PCR Master Mix Amount per Reaction 12.5 µl DAD629 (nptii) (20mM) 1.0 µl DAD630 (nptii) (20mM) 1.0 µl nuclease free water 8.5 µl 23 µl *Use 23 µl of the above mixture plus 2 µl of DNA for a total reaction volume of 25 µl *The ideal annealing temperature for the nptii primers is 60 C so use program KZ60 on the thermal cycler Step 1: 94 C: 5 minutes Step 2: 94 C: 1 minute Step 3: 62 C: 1 minute Step 4: 72 C: 1 minute, 30 seconds Step 5: Repeat steps 2 through 4, 30x for a total of 31 cycles Step 6: 72 C: 4 minutes Step 7: Hold at 4 C Fig. 2 Lane 1: 100 bp DNA ladder; lane 2 = positive control, lane 3 = negative control, lanes 4-33 = population segregating for the nptii gene *The nptii band should be 229 bp