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page 1 Simple Fool’s Guide P C R THE SIMPLE FOOL’S GUIDE TO PCR version 2.0 Tue

page 1 Simple Fool’s Guide P C R THE SIMPLE FOOL’S GUIDE TO PCR version 2.0 Tuesday, October 29, 2002 Version 2 compiled by: Steve Palumbi Department of Zoology and Andrew Martin Kewalo Marine Laboratory Sandra Romano University of Hawaii W. Owen McMillan Honolulu, HI 96822 Ligaya Stice (808) 539-7300 Gail Grabowski (808) 599-4817 (FAX) P C R page 2 Simple Fool’s Guide P C R TABLE OF CONTENTS Table of Contents ............................................................................2 Introduction to S.F.G.........................................................................3 DNA Extraction For PCR ....................................................................4 General Extraction Protocol for Total DNA..........................................................4 Variations on the Extraction Protocol...................................................................5 The Cycle .....................................................................................7 The Basic Cycle...................................................................................................7 Variations in the Cycle.........................................................................................8 PCR Protocols................................................................................9 Hy"gene" ......................................................................................................9 Protocols ......................................................................................................9 Frequently used solutions. ...........................................................................10 Double-Strand DNA Amplifications ..............................................................11 Double strand problems. ................................................................11 Single-Strand DNA Amplifications.................................................................12 Single strand problems...................................................................13 Double-Strand mRNA Amplifications.............................................................14 Sequencing protocols..................................................................... 15 From single strand PCR products.......................................................................15 From double strand PCR products......................................................................17 Solid phase stripping with biotinylated primers...................................................18 Lambda exonuclease method.............................................................................20 Running the Sequencing Gel............................................................. 20 PCR Primers................................................................................ 22 Making them ...............................................................................................22 Purifying them.............................................................................................23 Mitochondrial primers ..................................................................................25 12s RNA Primers...........................................................................26 16s RNA Primers...........................................................................28 Cytochrome oxidase II Primers. ......................................................29 Cytochrome b Primers....................................................................32 D-loop Primers ..............................................................................35 Cytochrome Oxidase II Primers ......................................................37 ATPase 6 Primers.........................................................................37 Nuclear Gene Primers .................................................................................38 bindin............................................................................................38 int.................................................................................................39 creatine kinase. .............................................................................40 Vector primers ............................................................................. 41 MtDNA maps................................................................................ 42 Contributors................................................................................ 43 References to Complete mtDNA Sequences ........................................... 44 Selected References to PCR.............................................................. 44 Solution Appendix ......................................................................... 45 Abbreviations .............................................................................. 46 page 3 Simple Fool’s Guide P C R INTRODUCTION TO S.F.G. The Simple Fool’s Guide to PCR , a collection of PCR protocols and oligonucleotide primers, is an attempt to promote sharing of PCR protocols and primer sequences from different gene regions, so that redundant (and costly) effort in the refinement of PCR techniques and the design and making of primers is not wasted. Although PCR is extremely fast and easy, there are (in our minds) a number of mysterious manifestations and inconsistencies that rear their ugly heads when working with new species. We hope that while we might have encountered and solved some mysteries that others are running across, other workers may have solved ones with which we need help. Please keep in mind that these protocols are ones that we have had consistent success using on various taxa in our lab, but that does not mean that they will work perfectly with the DNA with which you are working or that other methods are not better. It is also not an attempt to make a complete reference for PCR techniques and primers. Rather it is an attempt to collect easy PCR techniques and primers that just about anyone can use with success. We have gathered some of these methods from colleagues across the country. Some have been scribbled on beer-soaked coasters in dark Berkeley bars. Some have resulted from midnight airport rendezvous. We have found in general that people have been delightfully open and generous with their technique development and primers. One of our goals is to promote continued openness with this guide. The compilation of this “guide” is not an altruistic act. We are asking that other labs share their experiences with us. If you or your lab uses any of the techniques or primers that we have gathered together in this guide we ask that you let us know what works with what under what conditions. Subsequent versions of SFG (if there are any) will contain such information as is given unto us along with your name and affiliation in the “Contributors” section. PCR has largely been developed by workers at Perkin Elmer Cetus and we wish to thank them collectively for all their help. We also would like to point out that Perkin Elmer Cetus publishes a newsletter called “Amplifications” which often has PCR protocols and suggestions that are extremely useful. We start in the spirit of simple foolishness by describing DNA extraction, the basic PCR cycle, and some general guidelines we have found useful. Suggestions (like those in the “variations” section) are followed up in greater detail below. Good luck. © X We reserve no rights for this document or its contents. Copy, maim, or spit on this document as you like. page 4 Simple Fool’s Guide P C R DNA EXTRACTION FOR PCR Because PCR uses sequence specific primers, it is possible (in theory) to amplify any portion of the mitochondrial or nuclear genome using total genomic extracts. These extracts may be obtained from fresh or wet/dry preserved tissues. Protocols for the extraction of total DNA from any of these tissue sample types are generalizable to most sample types after the performance of a few initial steps specific to each type. Presented below, in detail explicit enough for even your mother to follow, is a description of a general method for extracting total DNA from fresh tissue samples. Additions/deletions to this protocol for preserved samples are noted following “Mom’s recipe for homemade DNA extracts”. We welcome comments on other methods that may be superior to these. A. General Extraction Protocol for Total DNA This procedure has worked well for fresh tissue samples obtained from animals representing a variety of phyla from corals to urchins to sharks to even mammals. 1. Procure/dissect out roughly 0.1 - 0.5 grams of the the desired tissue type and place in a 1.5 ml micro-centrifuge tube. DON’T BE GREEDY (it is easier to use too much tissue than too little). NOTE: If you are working with a tissue that is especially DNA dense, such as sperm, do not start with “generous” quantities of gonads or cells. Obtain sparing samples and wash them in a water/sea water/Ringer’s etc. solution before putting them in the Lysis Buffer. 2. Add an approximately equal volume of Lysis Buffer to the tissue swatch. Lysis Buffer: 100mM EDTA 10mM Tris (pH 7.5) 1% SDS 3. Macerate/grind the tissue. We use a “micro-pestle” which is made by pouring plastic casting resin (used to repair surfboards, easily available in most labs in Hawaii) into a micro-centrifuge tube and letting it harden with a stiff rod inserted into it for a handle. Insert the pestle gently into the microfuge tube and rotate it. Make about three “passes” at the tissue (or until it appears milky). NOTE: Samples that are too DNA dense at this point have an extremely high viscosity and exhibit impressive viscoelasticities (= snotlike for non-biomechanics) after maceration. This physical property hinders the effectiveness of subsequent separation procedures so dilute such samples in Lysis buffer and macerate until the viscoelastic behavior of the solution is greatly reduced. 4. Microcentrifuge this solution for approx. 3 min at 14,000 rpm. 5. Decant the supernatant and save. NOTE: If you are working with more than one preparation its a good idea to keep the samples on ice between performing steps of the extraction procedure. This precaution may slow down any enzymatic digestion of the DNA that may occur before it is isolated. 6. Add an approximately equal volume of buffer-equilibrated phenol to the decanted supernatant and invert gently. 7. Microcentrifuge for 10 min at 14,000 rpm. 8. After centifuging, the DNA should be suspended within the upper aqueous layer. Immediately under this layer and above the bottom phenol layer there may be a whitish layer that contains proteins and carbohydrates. Pipet off only the topmost layer and save. If your sample consists of only two layers remove all of the upper layer. page 5 Simple Fool’s Guide P C R NOTE: Occasionally, when DNA density is high, the DNA containing layer is heavier than the phenol. Therefore the bottom layer after centrifugation will contain the DNA. For this reason always save the bottom layer after decanting the upper layer. (It will become obvious in subsequent steps if you’ve decanted the phenol layer and should be working with the bottom layer instead.) 9. Add an approximately equal volume of 1:1 phenol/chloroform to the decanted liquid and invert gently. If you are working with the DNA containing layer it will not mix with the phenol/chloroform. If it does mix, you’ve got the wrong layer, so try adding the phenol/chloroform to the bottom layer from step 8. 10. Microcentrifuge for 5 min at 14,000 rpm. 11. Pipet off and save the clear/whitish DNA containing layer off the top of the phenol/chloroform. 12. Add an approximately equal volume of chloroform to the decanted liquid and invert gently. 13. Microcentrifuge for 3 min at 14,000 rpm. 14. Pipet off the top layer into well labeled, pre-weighed microcentrifuge tubes. These will be the final sample containers. 15. Weigh each sample and determine the volume of each by assuming 1 gram of the liquid = 1 ml. 16. Add a volume of 7.5 M NH4OAc equal to 1/2 sample volume. 17. Add to this solution either (1) enough 2-propanol to make the solution 50% 2-propanol, or (2) enough ethanol to make the solution 66% ethanol. 18. Invert gently and let the solutions sit for at least 10 min at room temperature. NOTE: The DNA will precipitate out of solution in this step as will unwanted salts uploads/Geographie/ pcr-guide.pdf