General PCR protocol Page 1 of 5 This protocol describes how to: - do a good PCR - set up a new PCR protocol - do trouble shooting And includes a sheet for noting your PCR mix components and amounts and PCR programm. Before performing a PCR Performing a PCR is very easy, performing a good PCR with general eubacterial primers isn't!!! You easily can have contamination ruining your experiment. Therefore: - Work only in the DNA lab, pipet the premix only in the special premix-box, pipet the template to the premix only in the template-box. Before starting check whether the UV light has been on during the night. Check whether there are still sufficient 20 and 200 µl tips. The tips to be used must have a cotton plug. Automatic pipets may never be removed from the boxes. Put dirty tips immeadeatly in the bins outside the boxes. When you have used the boxes, please note date etc. in the book near it. Remove waste after use. - Wear a clean lab coat (especially check the sleeves) and clean gloves and change gloves frequently. While hand gloved avoid contact with materials which you don't necessarly have to touch (for example chairs and tables). Otherwise, change gloves!!! Also fill boxes with tips or tubes etc while hand gloved. - Use only heat sterilized and disposable tips and tubes. Clean and sterilized tips and tubes are found on top of the refrigerator. Empty boxes etc can be put on the table left of the door through which you enter the lab. At this moment we don't have a population of goblins in the lab, so it is wise to also fill and sterilize the boxes etc yourself. Sterilize by heat, 4h at 120oC in the oven in Coen's lab. - Store materials you use for PCR (primers etc), with the exception of the template!!!!, in a special Boehringer box, in the primer box drawer in the freezer in the DNA lab. Store template in your own drawer in the freezer. NEVER touch tubes etc from the primer box drawer after touching boxes/tubes with DNA template. Performing the PCR Wait!! - Check the premix- and template boxes, are they and their environments clean, has the UV light been on at night? - Do you have sterile tubes and tips? 1. Make a list of the premix (amounts of primers, buffer, dNTPs, enzyme, additions (BSA, DMSO)) which you are planning to make. Run always a blank and one positive control (DNA from a strain). For x environmental DNA samples calculate the amount for x + 3 reactions (pos. and neg. control, + 1 to compesate for small pipetting errors). Use the sheet in this document. general pcr protocol.doc October 24, 2013 General PCR protocol Page 2 of 5 In general 24 µl premix is made per reaction, it contains: 1 µl forward primer (10 µM) 1 µl reverse primer (10 µM 1 µl dNTPs (0.2-0.4 mM) 2.5 µl buffer enzyme, possible DMSO and/or BSA it is filled up with MilliQ water to 24 µl per reaction. 2. Put on gloves before you take the PCR mix components or master mix out of the freezer box. Place all the components in the PREMIX box, so that they can become soluble. REMEMBER TO CHANGE GLOVES FREQUENTLY. 3. Place the template DNA on ice on the table, NOT in one of the PCR-boxes. CHANGE GLOVES. 4. Prepare the premix, like you have calculated. As last, add the enzyme. 5. Gently vortex the mixture. 6. Put it 15 minutes under the UV lamp in the premixbox. 7. Centrifuge it for 10 seconds. 8. Divide the mix over the PCR vials (24 µl in one vial). Close vial immeadeatly after filling. 9. Take the tubes out of the PCR premix box. 10. Clean the boxes and their environment. Inscribe into the book. 11. On the table add 1.0 µl DNA template to the PCR vial. Close vial immeadetly after filling. 12. Vortex and centrifuge shortly. 13. Place the samples in the thermocycler and start your PCR programm. This programm should contain: - initial denaturation step: 4 min 94oC - Over 25 cycles of denaturation (at 92oC - 94oC for 0.5 to 1 min), annealing (dependent on primer, see below) and extension (68 - 72oC for 1 to 2 minutes, depending on length of PCR product) - final elongation; 5 min. 72oC. - a cooling step to 4oC max 15 min. 14. After PCR, check 5 µl of the mix on a 1.5% agarose TAE gel. Store remainder at -20oC in special PCR tube boxes. general pcr protocol.doc October 24, 2013 General PCR protocol Page 3 of 5 Developing a specific PCR protocol For every environmental sample or primer pair you probably have to develop a new PCR protocol. For some environments (landfill, forest soils) and bacterial groups (ammonia oxidizers) this has already be done. Composition of the PCR mix: Some suggestions, to test: - DNA template often contains PCR inhibitors, dilution of template and/or addition of BSA might help. - DNA polymerases have different sensitivities to inhibitors, testing different polymerases might help. - For better annealing of primers to DNA template try DMSO. PCR programm: -To determine a suitable annealing temperature, calculate the melting temperature (Tm) of your primers: Tm = 4 x number of G and C in primer + 2 x number of A and T in primer. A suitable annealing temperature to test first is the average Tm of both primers minus 5. Differences in Tm of primers should preferably be less than 5 degrees, otherwise take as first annealing temperature to test the lowest Tm minus 5. - Try different numbers of cycles. - Decrease denaturation and/or extension temperature to save the polymerase from inactivation. Suggestion: When developing a new protocol for a new environmental sample, include also a reaction in which you mix the environmental DNA template (1 µl) with the positive control (1 µl). This is an internal control, to check for reaction inhibiting substances. general pcr protocol.doc October 24, 2013 General PCR protocol Page 4 of 5 Trouble shooting A. You did not get a product in your environmental sample. 1. The positive control was negative. Either you forgot something or one of your mix components is not OK. Repeat the experiment. Still no product? Check with others about their results, use new components. 2. The positive control was positive. Probably there are inhibitors in the reaction. Repeat the PCR, include a reaction in which environmental DNA and DNA used as positive control are combined (internal control). Still no product? - dilute the environmental sample 1/10, 1/100 and 1/1000 and perform a PCR. Again prepare per dilution also an internal control. - if there was no BSA and/or DMSO in your mixture examine the effect of adding these compounds - perform an extra cleaning of your DNA sample. B. You also got a product in your negative control. How much was it, compared to your environmental samples? Many polymerases contain some DNA, which can give a positive reaction in the negative control, especially with a large number of cycles runned. If the contamination is limited, you may not really have a problem. Remember that many environmental samples contain inhibitors, but your negative control doesn't. Thus contaminant DNA in the blanc is relatively easier amplified!!! Check all your samples by DGGE to judge this. If you have a large contamination, repeat the experiment once and if required dispose the components you used and use new ones. general pcr protocol.doc October 24, 2013 General PCR protocol Date: Title: PCR machine: Page 5 of 5 Labjournal: Programm: Temperature oC Initial denaturation Cycle programm Time ' " Cycle number: Denaturation Annealing Extension oC oC oC ' ' ' " " " Final extension oC ' " Premix forward primer = reverse primer = dNTPs BSA DMSO buffer ........ Master mix MQ water Enzyme = 1x Premix Total Sample (name/code) 1 negative control 2 positive control 3 4 5 6 7 8 9 10 11 12 13 14 15 16 general pcr protocol.doc µl µl µl µl µl µl µl µl µl µl samples µl µl µl µl µl µl µl µl µl µl µl µl Storage box: 17 18 19 20 21 22 23 24 October 24, 2013 x+3=