Fungal gdna Mini Kit. User Guide. Cat No: XG XcelGen Ver.: 0.2/13 Revised Protocol

Transcription

1 User Guide Cat N: XG XcelGen Ver.: 0.2/13 Revised Prtcl

2 Table f Cntents Intrductin Overview Strage and Stability Kit Cntents Befre Starting Fungal gdna Miniprep Prtcl A. Dry Specimens B. F resh/frzen Specimens Vacuum/Spin Prtcl Trubleshting Guide Limited Use and Warranty

3 Intrductin Fungal gdna Miniprep Kits allw rapid and reliable islatin f high-quality ttal cellular DNA frm a wide variety f Fungal species and tissues. Up t 100 mg f wet tissue (r up t 50 mg dry tissue) can be prcessed within shrt time. The system cmbines the reversible nucleic acid-binding prperties f XcelGen matrix with the speed and versatility f spin clumn technlgy t eliminate plysaccharides, phenlic cmpunds, and enzyme inhibitrs frm fungal tissue lysates. Purified DNA is suitable fr PCR, restrictin digestin and hybridizatin techniques. There are n rganic extractins thus reducing plastic waste and hands-n time t allw multiple samples t be prcessed in parallel. Overview If using the Fungal gdna Miniprep Kit fr the first time, please read this bklet t becme familiar with the prcedures befre beginning. Dry r fresh fungal tissue is disrupted and then lysed in a specially frmulated buffer cntaining detergent. Prteins, plysaccharide and cellular debris are subsequently precipitated. Binding cnditins are then adjusted and the sample is applied t a DNA spin-clumn. Tw rapid wash steps remve trace cntaminants such as residual plysaccharide, and pure DNA is eluted in water r lw inic strength buffer. Purified DNA can be directly used in dwnstream applicatins withut the need fr further purificatin. Strage and Stability All cmpnents f the Fungal gdna Miniprep Kit are stable fr at least 12 mnths when stred at 22 C-25 C. During shipment r strage in cl ambient cnditins, precipitates may frm in Buffer FG1,FG2 and Buffer BL. It is pssible t disslve such depsits by warming the slutin at 50 C. RNase A shuld be stred at 4 C. 02

4 Kit Cntents Prduct XG XG DNA Clumns 2 ml Cllectin Tubes Buffer FG1 Buffer FG2 Buffer BL RNase A DNA Wash Buffer Elutin Buffer User Guide ml 1.0 ml 1.8 ml 30 l 2 ml 1.5 ml ml 10 ml 22 ml 270 l 15 ml 15 ml 1 Befre Starting Prepare all cmpnents and get all necessary materials ready by examining this instructin bklet and becme familiar with each steps. Imprtant Pre-heat FG1 and Elutin Buffer t 65ºC. Dilute Wash Buffer Cncentrate with ethanl as fllws and stre at rm temperature. Add 8 ml (XG ) r 60 ml (XG ) abslute (96%-100%) ethanl t each bttle. Chse the mst apprpriate prtcl t fllw. Prcedures are described fr each f dried and fresh (r frzen) specimens. A. Dry Specimens : Fr prcessing 50 mg pwdered tissue. Yield is sufficient fr several tracks n Suthern assay. B. Fresh r Frzen : Fr prcessing 100 mg fresh (r frzen) pwdered tissue. Yield is similar t A. 03

5 Fungal gdna Mini Kit Prtcl A. Dry Specimens Materials supplied by users: Centrifuge capable f at least 10,000g Nuclease-free 1.5 ml r 2.0 ml micrfuge tubes Waterbath equilibrated t 65 C Equilibrate sterile ddh2o water at 65 C Abslute (96%-100%) ethanl Paper twels This is the mst rbust methd fr islatin f ttal cellular (mitchndrial. chlrplast, and genmic) DNA. Yields are usually sufficient fr several tracks n a Suthern blt fr RFLP mapping. Drying allws strage f field specimens fr prlnged perids f time prir t prcessing. Samples can be dried vernight in a 45 C ven, pwdered, and stred dry at rm temperature. T prepare dried samples place ~50 mg f dried tissue int a micrfuge (2 ml tubes are recmmended fr prcessing f >50 mg tissue) tube and grind using a pellet pestle. Dispsable Kntes pestles wrk well. Fr critical wrk such as PCR and clning, pestles are best used a single time then saked in a dilute bleach slutin immediately after use until cleaning. Dispsable pestles may be autclaved several times. Fr standard Suthern analysis, the same pestle can be reused several times t grind multiple tissue samples by rinsing with ethanl and wiping the surface clean between samples. A fine pwder will ensure ptimal DNA extractin and yield. Prcess in sets f fur t six tubes until Step 2 befre starting anther set. 1. Take mg pwdered dry tissue add 600 l Buffer FG 1, Vrtex vigrusly t mix. 2. Incubate at 65 C fr 30 min. Mix sample twice during incubatin by inverting tube. Optinal: If necessary, add 5 remve the RNA. l f RNase A int the lysate befre incubatin t 3. Add 600 l chlrfrm/isamyl alchl (24:1) and vrtex t mix. Centrifuge at 10,000g fr 10 min. 4. Carefully aspirate 300 l supernatant t a new 1.5 ml micrfuge tube making sure 04

6 05 nt t disturb the pellet r transfer any debris. 5. Adding 150 l Buffer FG 2 fllwed by 300 l abslute ethanl and vrtex t btain a hmgeneus mixture. A precipitate may frm upn additin f ethanl; it will nt interfere with DNA islatin. 6. Add 400µl f Buffer BL int the spin clumn (Prvided), incubate at rm temperature fr 2 min, centrifuge at rpm fr 2 min and discard the flw thrugh. The clumn is ready and wrk well fr binding DNA. 7. Apply the entire sample (including any precipitate that may have frmed) t a DNA clumn placed in a 2 ml cllectin tube (supplied). Centrifuge the clumn at 10,000 g fr 1 min t bind DNA. Discard bth the 2 ml cllectin tube and the flwthrugh liquid. 8. Transfer clumn t a secnd cllectin tube and wash by adding 650 l DNA Wash Buffer diluted with abslute (96%-100%) ethanl. Centrifuge at 10,000g fr 1 min and discard the flw-thrugh liquid. Nte: Wash Buffer Cncentrate must be diluted with abslute (96%-100%) ethanl prir t use. Fllw directins n label. 9. Repeat wash step with an additinal 650 l DNA Wash Buffer. Centrifuge at 10,000g fr 1 min. 10. Centrifuge empty clumn 2 min at maximum speed t dry. This step is critical fr remving residual ethanl that may therwise be eluted with DNA and interfere with dwnstream applicatins. 11. Transfer clumn t a clean 1.5ml micrfuge tube. Apply 100 l Elutin Buffer (r sterile deinized water) pre-warmed t 65 C and incubate at rm temperature fr 3 t 5 min. Centrifuge at 10,000g fr 3 t 5 min t elute DNA. Smaller vlumes will significantly increase DNA cncentratin but give lwer yields. Use f mre than 200 l f buffer fr elutin is nt recmmended. 12. Repeat Step 11 with an additinal 100 l f Elutin Buffer. This may be perfrmed using anther 1.5ml tube t maintain a higher DNA cncentratin in the first eluate. Nte: T increase DNA cncentratin, add elutin buffer and incubate the clumn at 60 C-65 C fr 5 min befre elutin. 13. Ttal DNA yields vary depending n type and quantity f sample. Typically, g DNA with a A 260/A 280 rati f can be islated using 50 mg dried tissue.

7 B. Fresh r Frzen Specimens Materials t be prvided by user: Micrcentrifuge capable f 10,000g Nuclease-free micrfuge tubes Waterbath equilibrated t 65 C Equilibrate sterile ddh2o water at 65 C Abslute (96%-100%) ethanl Liquid nitrgen fr freezing/disrupting samples Paper twels Nte: Use extreme cautin when handling liquid nitrgen. This prtcl is suitable fr mst fresh r frzen tissue samples, allwing efficient recvery f DNA. Hwever, due t the tremendus variatin in water and plysaccharide cntent f varius fungi, sample size shuld be limited t 100 mg. The methd islates sufficient DNA fr several tracks n a standard Suthern assay. T prepare samples, cllect tissue in 1.5ml r 2ml micrfuge tube and freeze by dipping in liquid nitrgen with a pair f tweezers t fill the tube. Grind the tissue using dispsable pellet pestles. Alternatively, ne can allw liquid nitrgen t evaprate and then stre samples at -70 C fr later use. Fr critical wrk such as PCR and clning, pestles are best used a single time then saked in a dilute bleach slutin immediately after use until clean. Dispsable pestles may be autclaved several times. Fr standard Suthern analysis, the same pestle can be reused several times t grind multiple tissue samples by rinsing with ethanl and carefully wiping the surfaces clean between samples. 1. Cllect grund fungal tissue (start with 100 mg) in a micrfuge tube and immediately add 500 l Buffer FG 1, Vrtex vigrusly t mix. 2. Incubate at 65 C fr 15 min. Mix sample twice during incubatin by inverting tube. Optinal: If necessary, add 5 l f RNase A int the lysate befre incubatin t remve the RNA. 3. Add 800 l chlrfrm/isamyl alchl (24:1) and vrtex t mix. Centrifuge at 10,000g fr 5 min. 4. Carefully aspirate 300 l supernatant t a new 1.5 ml micrfuge tube making sure nt t disturb the pellet r transfer any debris. 06

8 5. Adding 150 l Buffer FG 2 fllwed by 300 l abslute ethanl and vrtex t btain a hmgeneus mixture. A precipitate may frm upn additin f ethanl; it will nt interfere with DNA islatin. 6. Add 400µl f Buffer BL int the spin clumn (Prvided), incubate at rm temperature fr 2 min, centrifuge at rpm fr 2 min and discard the flw thrugh. The clumn is ready and wrk well fr binding DNA. 7. Apply the entire sample (including any precipitate that may have frmed) t a DNA clumn placed in a 2ml cllectin tube (supplied). Centrifuge the clumn at 10,000g fr 1 min t bind DNA. Discard bth the 2ml cllectin tube and the flwthrugh liquid. 8. Transfer clumn t a secnd cllectin tube and wash by adding 650 l DNA Wash Buffer diluted with abslute (96%-100%) ethanl. Centrifuge at 10,000g fr 1 min and discard the flw-thrugh liquid. Nte: Wash Buffer Cncentrate must be diluted with abslute (96%-100%) ethanl prir t use. Fllw directins n label. 9. Repeat wash step with an additinal 650 l DNA Wash Buffer. Centrifuge at 10,000g fr 1 min. 10. Centrifuge empty clumn 2 min at maximum speed t dry. This step is critical fr remving residual ethanl that may therwise be eluted with DNA and interfere with dwnstream applicatins. 11. Transfer clumn t a clean 1.5ml tube. Apply 100 l Elutin Buffer (r sterile deinized water) pre-warmed t 65 C and incubate at rm temperature fr 3 t 5 min. Centrifuge at 10,000g fr 3 t 5 min t elute DNA. Smaller vlumes will significantly increase DNA cncentratin but give lwer yields. Use f mre than 200 l f buffer fr elutin is nt recmmended. 12. Repeat Step 11 with an additinal 100 l f Elutin Buffer. This may be perfrmed using anther 1.5 ml tube t maintain a higher DNA cncentratin in the first eluate. Nte: T increase DNA cncentratin, add elutin buffer and incubate the clumn at 60 C-65 C fr 5 min befre elutin. 13. Ttal DNA yields vary depending n type and quantity f sample. Typically, g DNA with a A 260/A 280 rati f can be islated using 100 mg fresh tissue. 07

9 Vacuum/Spin Prtcl Nte: Please read thrugh previus sectins f this manual befre using this prtcl. 1. Prepare wet r dry samples by fllwing the standard Prtcl in previus sectins until lading Buffer BL t DNA clumn. 2. Prepare the vacuum manifld accrding t manufacturer s instructins and cnnect the V-Spin clumn t the manifld. 3. Lad the DNA/FG2/Ethanl slutin t the ready clumn. 4. Switch n vacuum surce t draw the sample thrugh the clumn and turn ff the vacuum. 5. Wash the clumn by adding 650 l DNA wash buffer. Draw the wash buffer thrugh the clumn by turning n the vacuum surce. Repeat this step with anther 650 l DNA wash buffer. 6. Assemble the clumn int a 2 ml cllectin tube and transfer the clumn t a micr centrifuge. Spin 1 min t dry the clumn. 7. Place the clumn in a clean 1.5 ml micrfuge tube and add 100 l Elutin Buffer r deinized water. Stand fr 1-2 min and centrifuge 1 minute t elute DNA. L Fig: Agarse gel analysis f Fungus gdna purified with XcelGen Fungus gdna mini Kit. Lane 1 : gdna islated frm Hyphae. Lane 2 : gdna islated frm Mushrm. Lane 3 : gdna islated frm Filamentus fungus. Lane 4 : gdna islated frm Cnidia. Lane L : Hind III DNA ladder 08

10 Trubleshting Guide Prblem Pssible reasn Suggestins Clgged well Carry-ver f debris. Fllwing precipitatin with chlrfrm / Isamyl alchl, make sure n particulate material is transferred. DNA pellet nt cmpletely disslved befre applying sample t clumn. Sample t viscus In prtcls A and B, ensure that DNA is disslved in water befre adding Buffer FG 2 and ethanl. This may need repeated incubatin at 65 C and vrtexing. In prtcl C, d nt exceed suggested amunt f starting material. Alternatively, increase amunts f Buffers FG 1 and FG 2 and use tw r mre clumns per sample. Lw DNA yield Prblems in dwnstream applicatins Incmplete disruptin f starting material. Pr lysis f tissue. DNA remains bund t clumn. DNA washed ff. Salt carry-ver. Ethanl carry-ver Fr bth dry and fresh samples, btain a fine hmgeneus pwder befre adding Buffer FG1. Decrease amunt f starting material r increase amunt f Buffers FG 1, chlrfrm / Isamyl alchl and FG 2. Increase elutin vlume t 200 l and incubate n clumn at 65 C fr 5 min befre centrifugatin. Dilute Wash Buffer Cncentrate by adding apprpriate vlume f abslute ethanl prir t use. DNA Wash Buffer must be at rm temperature. Fllwing the secnd wash spin, ensure that the clumn is dried by centrifugatin fr 2 min at maximum speed. 09

11 Limited Use and Warranty This prduct is intended fr in vitr research use nly. Nt fr use in human. This prduct is warranted t perfrm as described in its labeling and in XcelGens literature when used in accrdance with instructins. N ther warranties f any kind, express r implied, including, withut limitatin, implied warranties f merchantability r fitness fr a particular purpse, are prvided by XcelGen. XcelGens sle bligatin and purchaser s exclusive remedy fr breach f this warranty shall be, at the ptin f XcelGen, t replace the prducts, XcelGen shall have n liability fr any direct, indirect, cnsequential, r incidental damage arising ut f the use, the results f use, r the inability t use it prduct. Fr technlgy supprt r fr mre prduct infrmatin, please visit ur website at 10

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