Buccal or eggshell swabs

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1 Buccal r eggshell swabs DNA Extractin Prcedure December 2013 Buccal r eggshell swabs It s imprtant t test befrehand the efficacy f this prtcl n a few samples (nt all f them at nce), just in case yu have t adjust yur prtcl (lysis time, elutin vlume, ). 1. DNA extractin 1.1. Genmic DNA frm Tissue NucleSpin Tissue Machery-Nagel Cnsumables ml micrcentrifuge tubes fr sample lysis and DNA elutin - Dispsable tips Labratry equipment - Manual pipettrs - Centrifuge fr micrcentrifuge tubes - Vrtex mixer - Heating-blck fr incubatin at 70 C - Equipment fr sample disruptin and hmgenizatin - Persnal prtectin equipment (lab cat, glves) Reagents % Ethanl - NucleSpin Tissue Kit : Lysis Buffer T1 Buffer B1 Buffer B2 Wash Buffer (BW) Wash Buffer B5 (cncentrate) Elutin Buffer BE Prteinase K (lyphilized) Prteinase Buffer PB Strage cnditins and preparatin f wrking slutins Clean the bench tp with alchl befre and after setting up extractins. Use the filter-plugged tips t avid cntaminatin f samples and reagents. All kit cmpnents can be stred at RT and are stable up t ne year. If a white precipitate ccurs in buffer T1, B1 r B3, it can be easily disslved by incubating the bttle at C befre use. Befre starting the extractin prtcl, prepare the fllwing : Prepare the Lysis Buffer B3 : transfer the ttal cntents f Buffer B1 t Buffer B2 and mix well, it s Buffer B3. 1

2 Buccal r eggshell swabs DNA Extractin Prcedure December 2013 Prepare the Wash Buffer B5 : add the indicated vlume f ethanl ( %)t the Wash Buffer B5 cncentrate. Prepare the Prteinase K Slutin : add the indicated vlume f Prteinase K Buffer PB t disslve lyphilized Prteinase K. This slutin is stable at -20 C fr up t 6 mnths. Prducts 10 Preps 50 Preps 250 Preps Lysis Buffer B3 Add Buffer B1 (6 ml) t Buffer B2 (1.5 ml) Add Buffer B1 (12 ml) t Buffer B2 (3 ml) Add Buffer B1 (60 ml) t Buffer B2 (15 ml) Wash Buffer B5 4 ml - Add 16 ml Ethanl 2 x 7 ml - Add 28 ml Ethanl t each bttle 2 x 40 ml - Add 160 ml Ethanl t each Prteinase K 6 mg Add 260 µl Prteinase buffer 30 mg Add 1.35 ml Prteinase buffer bttle 2 x 75 mg Add 3.35 ml Prteinase buffer t each vial Preparatin f the sample Set an incubatr r water bath t 56 C and 70 C. Preheat Elutin Buffer BE t 70 C. Place the dry swab in the micrtube. If the swab was stcked in ethanl, it needs t be cmpletely dried befre the DNA extractin. Incubate at 37 C fr abut 15 min with the cap slightly lsened, t evaprate the ethanl. If the swab was frzen, it needs t be cmpletely defrst befre the DNA extractin Lyse f the sample Add µl PBS (depending n the size f the swab) and 25 µl Prteinase K Slutin t the swab. Vrtex t mix. The sample shuld be cvered with the lysis slutin. Incubate at 56 C until cmplete lysis (1 t 3 hurs). Vrtex ccasinally r use a shaking incubatr. The lysis can be dne vernight as well DNA extractin After digestin, centrifuge at 1500 g fr 15 secnds t remve any cndensate frm the cap. Add ne vlume f Buffer B3 (400 r 600 µl depending n the size f the swab) and vrtex. Incubate at 70 C fr 10 min. Vrtex briefly. If insluble particles are visible, centrifuge 5 min at maximum speed and transfer the supernatant int a new micrcentrifuge tube. 2

3 Buccal r eggshell swabs DNA Extractin Prcedure December 2013 Add ne vlume % ethanl t each sample (usually, 400 r 600 µl depending n the size f the swab), vrtex t mix. In case f precipitates, lad the sample t the clumn regardless. Fr each sample, place ne clumn int a cllectin tube. Transfer 600 µl f the sample n the clumn. Centrifuge fr 1 min at g. Discard the flw-thrugh and place the clumn back int a new cllectin tube. Repeat this step if any lysis slutin remains. Always psitin tubes in the centrifuge in the same rientatin, i.e. cap facing utward, this will allw the pellet t remain glued t the same side f the tube during repeated centrifugatins and minimize the lss f DNA pellets. 1 st wash : Add 500 µl buffer BW. Centrifuge fr 1 min at g. Discard flw-thrugh and place the clumn back int a new cllectin tube. 2st wash : Add 600 µl Buffer B5 t the clumn and centrifuge fr 1 min at g. Discard flw-thrugh and place the clumn back int a new cllectin tube. Dry silica membrane by centrifuging the clumn fr 1 min at g. Elute DNA by placing the clumn int a new 1.5 ml micrcentrifuge tube and add µl pre-warmed buffer BE (70 C). Incubate at RT fr 1 min. Centrifuge 1 min at g. Stre at -20 C (See 2. DNA strage fr ther ptins) DNeasy Bld & Tissue Kit QIAGEN Cnsumables ml micrcentrifuge tubes fr sample lysis and DNA elutin - Dispsable tips Labratry equipment - Manual pipettrs - Centrifuge fr micrcentrifuge tubes - Vrtex mixer - Heating-blck - Equipment fr sample disruptin and hmgenizatin - Persnal prtectin equipment (lab cat, glves) Reagents % Ethanl - DNeasy Bld & Tissue Kit : Buffer ATL Buffer AL Buffer AW1 (cncentrate) Buffer AW2 (cncentrate) Buffer AE 3

4 Buccal r eggshell swabs DNA Extractin Prcedure December 2013 Prteinase K Strage cnditins and preparatin f wrking slutins Clean the bench tp with alchl befre and after setting up extractins. Use the filter-plugged tips t avid cntaminatin f samples and reagents. All kit cmpnents can be stred at RT and are stable up t ne year. If a white precipitate ccurs in buffer AL and ATL, it can be easily disslved by incubating the bttle at 56 C befre use. Buffer AW1 et AW2 are supplied as cncentrates. Befre using it fr the first time, add the apprpriate vlume f ethanl (96-100%) as indicated n the bttle and shake thrughly Preparatin f the sample Set an incubatr r water bath t 56 C. Place the dry swab in the micrtube. If the swab was stcked in ethanl, it needs t be cmpletely dried befre the DNA extractin. Incubate at 37 C fr abut 15 min with the cap slightly lsened, t evaprate the ethanl. If the swab was frzen, it needs t be cmpletely defrst befre the DNA extractin Lyse f the sample Add µl PBS (depending n the size f the swab) and 20 µl Prteinase K Slutin. Vrtex t mix. The sample shuld be cvered with the lysis slutin. Incubate at 56 C until cmplete lysis (1 t 3 hurs). Vrtex ccasinally r use a shaking incubatr. Samples can be incubated vernight as well DNA extractin After digestin, centrifuge at 1500 g fr 15 secnds t remve any cndensate frm the cap Vrtex the samples fr 15 s. Add 400 r 600 µl Buffer AL (depending n the size f the swab), vrtex vigrusly. Then add 400 r 600 µl f ethanl % (depending n the size f the swab) and mix again thrughly by vrtexing. Transfer 600 µl f the sample n the clumn. Centrifuge at g fr 1 min. Discard flw-thrugh. Repeat this step if yu have mre lysis slutin. Always psitin tubes in 4

5 Buccal r eggshell swabs DNA Extractin Prcedure December 2013 the centrifuge in the same rientatin, i.e. cap facing utward, this will allw the pellet t remain glued t the same side f the tube during repeated centrifugatins and minimize the lss f DNA pellets. 1 st wash : Add 500 µl buffer AW1. Centrifuge fr 1 min at g. Discard flw-thrugh and place the clumn back int a new cllectin tube. 2 nd wash : Add 500 µl buffer AW2. Centrifuge fr 3 min at g. Discard flw-thrugh and place the clumn back int a new cllectin tube. Remve the DNeasy Mini spin clumn carefully s that the clumn des nt cme int cntact with the flw-thrugh, since this will result in carryver f ethanl. Elute DNA by placing the clumn int a new 1.5 ml micrcentrifuge tube and add µl buffer AE. Incubate at RT fr 1 min. Centrifuge 1 min at g. Elutin with 100 μl increases the final DNA cncentratin in the eluate, but als decreases the verall DNA yield. Stre at -20 C (See 2. DNA strage fr ther ptins). 2. DNA Strage Accrding t the DNA extractin prtcls described abve, exclusively use buffers t elute DNA. Fr strage, DNA can be stred : - At -20 C as a wrking slutin t be used fr PCR amplificatin. - At -80 C fr lng term strage in GenTegra TM DNA Tubes. - At rm temperature, in GenTegra TM DNA Tubes fr lng term strage : the transparent matrix applied t the bttm f this tubes allws strage f DNA at rm temperature in a manner that preserves DNA integrity, quality and purity. The DNA samples are prtected frm hydrlysis, xidatin and micrbial grwth DNA applicatin in GenTegra TM DNA Tubes If the samples are frzen, incubate at 38 C fr 1 minute. Centrifuge at lw speed fr a few secnds. Apply µl f the samples in each GenTegra TM tube accrding t the manufacturer s prtcl and mix by pipetting up and dwn 6 times t slubilize the GenTegra TM matrix. 5

6 Buccal r eggshell swabs DNA Extractin Prcedure December Drying and strage f DNA in GenTegra TM DNA Tubes Put in the FastDryer. Ensure the tube hlder is inserted in the FastDryer. Place rack f tubes with lids ff r unsealed micrplate n the tp f the tube hlder. Ensure that the pwer crd in lugged in. Clse the fan lid. Press the red n/ff witch t perate the FastDryer. Leave n fr 16 hurs t dry the samples r vernight. When drying is cmplete, cap r seal the tubes/plates and stre at rm temperature (21-25 C) 2.3. DNA recvery frm GenTegra TM DNA Tubes When yu want t recver DNA, apply a vlume f mlecular bilgy grade water accrding t the guidelines in the manufacturer s prtcl (usually µl). Incubate at rm temperature (21 25 C) fr 15 minutes. Mix t slubilize the DNA. Cap the tubes and vrtex 1 minute r pipette up and dwn 10 times fr the micrplate. Fllwing the recvery, an aliqut f DNA may be remved fr use and the sample dried again. This prcedure may be repeated multiple time until a maximum f 75% f the riginal sample is remved. Fr example : if a 200 µl sample is applied int a tube, the vlume f the sample can t drp belw 50 µl (25 % f the riginal vlume). If it has t drp belw 50 µl, yu have t freeze it at -80 C. 3. Additinal remarks Silica membrane based kits are superir fr rutine extractins f mst taxa regarding a lt f criteria, including efficiency, sample purity, fragment length, handling time and material cst. Based n ur experience and previus studies, we recmmend either the use f these tw kits : Genmic DNA frm Tissue, NucleSpin Tissue (Machery-Nagel) r DNeasy Bld & Tissue Kit (QIAGEN). In the literature, yu can als find several studies using ne f these tw kits fr extracting DNA frm buccal r eggshell swabs. Here is a nn-extensive list : Chatfield M., Mler P., Richards-Zawacki C. et al., The Amphibian Chytrid Fungus, Batrachchytrium dendrbatidis, in Fully Aquatic Salamanders frm Sutheastern Nrth America, PLS ONE, vl.7,

7 Buccal r eggshell swabs DNA Extractin Prcedure December 2013 Gdman R., Miller D. and Arars Y., Prevalence f Ranavîrus in Virginia Turtles as Detected by Tail-Clip Sampling versus Oral-Clacal Swabbing, Nrtheastern Naturalist, vl. 20, pp , Hssack B., Adams M., Campbell Grant E. et al., Reptiles Lw Prevalence f Chytrid Fungus in Amphibians f U.S. Headwater Streams, Jurnal f Herpetlgy, vl. 44, pp , Maletzky A;, Kaiser R. and Mikulicek P., Cnservatin Genetics f Crested Newt Species Triturus cristatus and T. carnifex within a Cntact Zne in Central Eurpe: Impact f Interspecific Intrgressin and Gene Flw, Diversity, vl.2, pp , Martín-Gálvez D., Peralta-Sánchez J., Dawsn D. et al., DNA sampling frm eggshell swabbing is widely applicable in wild bird ppulatins as demnstrated in 23 species, Mlecular eclgy resurces, vl. 11, pp , Müller A., Lenhardt P., Theissinger K., Prs and cns f external swabbing f amphibians fr genetic analyses, Eurpean Jurnal f Wildlife Research, vl. 59, pp , Schulte U., Gebhard F., Heinz L. et al., Buccal swabs as a reliable nn-invasive tissue sampling methd fr DNA analysis in the lacertid lizard Pdarcis muralis, Nrth Western Jurnal f Zlgy, vl.7, pp , Wellbrck A., Bauch C., Rzman J. et al., Buccal swabs as a reliable surce f DNA fr sexing yung and adult Cmmn Swifts, J. Ornithl, vl. 48, pp. 5-45,

8 Buccal r eggshell swabs DNA Extractin Prcedure December