1. Collecting samples :

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1 1. Collecting samples : + Preservation of samples is very important to conserve DNA + Preservation solutions and methods: * aceton (50-100%) (flammable) * ethanol (90-100%) (flammable) * 2-propanol (flammable) * diethylether (flammable) * ethylacetate (flammable, harmful) * DESS (dimethyl sulphoxide (harmful!), disodium EDTA, and saturated NaCl) * drying samples freezing samples short heating 65 C? * formaline is not a good preservative for DNA research!!! + Amount of preservative : The amount of preservative in proportion to the amount of organism is important. 10:1, 20:1 are good, 3:1 is bad. Mixing! + Large animals : cut tissue in pieces (e.g. crab leg)

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3 2. Isolating DNA : + three options : kits - home made solutions - direct PCR kits expensive, fast, good results cheaper, takes more time, good results Expensive, very fast, no DNA left if fails or other genes needed + home made solution I use : CTAB * Reagents needed : + Washing buffer : 76 % ethanol and 10 mm ammoniumacetate + CTAB : (store at room temperature) 2 % (w/v) CTAB (hexadecyltrimethylammoniumbromide) 1,4 M NaCl 0,2 % (v/v) 2-mercaptoethanol 20 mm EDTA 100 mm Tris/HCl ph 8, µg/ml proteinase K : (add just before use!!!!) + 7,5 M ammonium acetate + RNAse A: 10 mg/ml (make DNAse free : boil 20 minutes at 100 C)

4 + home made solution I mostly use : CTAB * Method : - fresh animals : just rince it with destilled water. Fixated in aceton, ethanol,... rince twice with destilled water and leave 1 hour in destilled water, - bring the animal or some muscular tissue in a 2 ml tube with screwcap - add 500 µl CTAB and freeze 10 minutes - add ± 0,1 g glasbeads and 6 µl proteinase K (1 mg/100 µl) - beadbeat during 30 seconds at 5000 cycles/minute - incubate 2 hours (or more) at 60 C and shake regularly. (If the tissue is not completely dissolved, add 3 µl proteinase K and incubate overnight at 37 C) - add an 250 µl ammonium acetate 7,5 M and shake gently - centrifuge 10 minutes at rpm (room temperature!) - transfer the liquid into an other tube (pellet is precipitated protein) and add an equal volume cold isopropanol, mix and put 1 hour at room temperature - centrifuge 15 minutes at rpm (4 C) - remove supernatant - re suspend the pellet in 30 µl sterile water and add 1 µl RNAse (if removal of RNA is not necessary, go to #) - incubate 15 minutes at 60 C - add an equal volume isopropyl and put 15 minutes to 1 hour at room temperature - centrifuge 15 minutes at rpm (4 C) - remove supernatant - # add 1 ml washing buffer and put 30 minutes at room temperature - centrifuge 5 minutes at rpm (4 C) - remove supernatant (spin down the remaining drops of ethanol and suck it out with a fine pipette) - add 30 µl sterile water - use 1 to 5 µl from serial dilutions for PCR

5 Beadbeaters

6 + home made solution I used once recently: Alkaline lysis (Promising!!!!!!) * Reagents needed : - Alkaline lysisbuffer 2.5 ml 10% SDS (sodiumdodecylsulfaat) 5.0 ml 1N NaOH 92.5 ml sterile MQ water (Filtersterilize, prepare aliquots and store at ambient temperature and in dark conditions.) * Method : - put a small amount of tissue in an 500 µl tube containing 20µl alkaline lysisbuffer. - short spin to collect everything at the bottom. - heat for 15 at 95 C and cool. - add 180 µl sterile MQ water en mix gently. - centrifuge for 1 at maximum rpm. - use 1-5 µl for pcr.

7 + Possible problems : * Using complete animals : - stumach can contain other organisms - fungi, bacteria can stick to the surface * Dry samples (museum) : - air humidity can let fungi and bacteria grow on tissue * Badly preserved samples : - DNA partially or completely degraded + Optional : * Check quality of DNA on gel : * Determine the concentration of DNA (Nanodrop)

8 Nanodrop spectrophotometer

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10 + Storage of DNA : * Short term : 4 C (a few months) * Long term : -18 C * My experience is that frozen DNA needs to be defrosted for a few hours before use (I put it at 4 C the day before use). + Transport or shipping of DNA : * Can be done at roomtemperature (a few days) * Longer : dried (tube or on paper) or in ethanol

11 3. PCR :

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13 + Lots of difference between polymerases :

14 + Use protocol according to manufacturer! Reaction composition : - water - buffer - MgCl2 - dntp's - 2 primers - template DNA - polymerase Cycling protocol : - initial denaturation : 3' 94 C - 3 step cycling (30-45 x): - denaturation : C - annealing : C - extension: 1' 72 C

15 + variations in cycling : e.g. Step-down PCR (less mismatch primer) : Cycling protocol : - initial denaturation : 3' 94 C - 3 step cycling (5 x): - denaturation : C - annealing : C decrease each step 1 C - extension: 1' 72 C - 3 step cycling (40 x): - denaturation : C - annealing : C - extension: 1' 72 C

16 + Primer design : - length : nuciotides - G/C content : % - calculating melting temperature : Tm = 2 x (A+T) + 4 x (G + C) - avoid mismatches at 3' side (last 8-10 bases) - avoid 3 or more G and/or C at 3' side - avoid 3'-end T - degenerate primers : you can use T where T or C is needed you can use G where A or G is needed

17 + Primer design (continued) : - IUB codes : degenerate primers - designing reverse primer :

18 + Troubleshooting PCR : - double bands contamination primer mismatch (increase annealingtemp.) - smear degraded DNA too much starting template - no result degraded DNA primers don't fit not enough starting template - weak bands not enough starting template insufficient number of cycles

19 + Whole genome PCR kit : - amplifies the whole genome in fragments of bp - Multiple Displacement Amplification (MDA) technology, which carries out isothermal genome amplification utilizing a uniquely processive DNA polymerase capable of replicating up to 100 kb without dissociating from the genomic DNA template (Phi29 DNA polymerase). - can be interesting for samples with low amount of DNA : amplify the whole genome starting from 2 µl DNA, result is 50 µl amplified DNA that can be used for PCR. (Sample I tried : 50 --> 500 ng/µl DNA)

20 Thank You Andy Vierstraete University of Gent Belgium