Rotavirus Detection and Typing

Transcription

1 Appendix ii Rotavirus Detection and Typing Nucleic acid extraction and reverse transcription Virus Detection by PCR Rotavirus VP7, VP4, VP6 and NSP4 genotyping Version

2 Contents 1. Specimen Storage and Preparation of 10% Faecal Suspension 3 2. RNA Extraction Manual Boom Extraction Automated extraction: QIAxtractor (VX extraction kit, QIAGEN) 5 3. Random Priming RT 9 4. Rotavirus VP6-specific PCR Rotavirus VP6-specific real-time PCR (Taqman) Rotavirus NSP4-specific PCR Rotavirus genotyping PCRs G-Typing consensus PCR (VP7) G-Typing Multiplex PCR VP7 semi-nested consensus PCR (sequence typing) P-Typing consensus PCR (VP4) P-Typing multiplex PCR Oligonucleotide primer and probe sequences RNA extraction reagents Disposal of toxic waste Agarose-gel electrophoresis 25 NOTE: All risk assessments and COSHH assessments associated with the following procedures should be performed locally 2

3 1. Specimen Storage and Preparation of 10% Faecal Suspension Upon receipt, the specimens should be stored at 4 o C. Coat with a layer of glycerol if the specimens are not going to be processed within a week. Prepare 10-20% faecal suspensions in balanced salt solution (BSS) (Minimum Essential Medium or Medium 199) in 2ml screw cap tubes (Sarstedt). Add 200ul of liquid sample to 2 ml of BSS or a bacteriological loop full (the size of a garden pea) from semi-solid samples. Store the 10% faecal suspension at 4 o C until processing. Once all investigations have been carried out the 10% suspensions can be stored at -70 o C. The original faecal sample should be stored at 4 o C under a layer of glycerol. Avoid freezing and thawing the specimens repeatedly 3

4 2. RNA extraction 2.1 Guanidinium isothiocyanate (GTC)/ silica gel extraction of nucleic acid The 'Boom' method [Boom et al., 1990 J Clin Microbiol]. 1. Add 200ul of faecal suspension to 1ml of lysis buffer-l6 (see section 8) buffer and 20ul of size fractionated silica (see section 8) in a 1.5ml screw-capped microcentrifuge tube. RNase-free distilled water is included in each run to act as a negative control. Cell-culture grown SA11 or RRV should be used as a positive control Vortex for 10 sec and incubate at RT for 15 min. 3. Pellet by centrifugation (microcentrifuge) for 15 sec (13,000rpm), discard the supernatant. (Collect the supernatants for disposal of toxic waste, see Disposal of toxic waste; see section 9). 4. Wash the pellet with 1ml of lysis buffer L2 (see section 8), mix well by vortexing and pellet by centrifugation as before. Repeat with L2 buffer then similarly wash twice with 1ml 70% ethanol and 1ml acetone once 2 (store the wash fluids for disposal). 5. After removal of the acetone (perform carefully as pellets may become dislodged) centrifuge and place tube with lid open at 56 o C in a dry heating block for 5 minutes. 6. Add 49ul of RNase-free distilled water and 1ul of RNasin, vortex and incubate at 56 o C for 15 min to elute the nucleic acid from the silica. 7. Pellet by centrifugation at 13,000rpm for 4 min and extract the supernatant (avoid disturbing the silica. Recentrifuge if silica becomes resuspended). This can be stored in a new microfuge tube at 4 o C for 24h or -70 o C for longer. 1 An animal rotavirus is used as a positive control in order to monitor laboratory contamination. 2 It is important to resuspend the silica completely between wash steps. 4

5 2.2 QIAxtractor from QIAGEN Please refer to manufacturer s instructions for instrument set-up and calibration VXB (Binding Buffer) VXB Binding Buffer 100ml Store at room temperature VXB Binding Additive 1.14g Store at 4 C If a whole bottle of VXB will be used up in <1month, add all the additive to the VXB bottle. VXL (Lysis Buffer) Reconstitute Carrier RNA in 400ul Elution Buffer VXL Lysis Buffer 10ml Store at room temperature VX Proteinase K 1.1ml Store at 4 C Carrier RNA 210ul Store at 4 C 1. Prepare Lysis Plate by adding 200ul of faecal suspension (see section 1.) to each well. RNase-free distilled water is included in each run to act as a negative control. Cell-culture grown SA11 or RRV should be used as a positive control 3. The wells of whole columns need to be used, so if there are insufficient samples, then 200 ul of water should be added to the remaining wells in the column. If the whole of the plate is NOT used seal unused columns on the capture plate with an adhesive plastic plate seal 2. Turn on the QIAxtractor followed by the computer. 3. Select and double-click on the protocol required. 4. A map of the QIAxtractor platform layout is displayed on the screen. 5. The nucleic acid Capture Plate is positioned in area A1. a. Remove the Waste Sink from the left hand side of area A1. b. On the bench, turn the Sink upside down and position over Silicone Mat. 3 An animal rotavirus is used as a positive control in order to monitor laboratory contamination. 5

6 c. Line up the mat and Sink and apply downward pressure to insert the Silicone Mat. d. Smear a small amount of silicone grease on the Sink O ring. e. Insert the Sink into A1 with the nozzle inserted into the vacuum chamber. f. Place the Transfer Carriage on top of the Silicone Mat and Waste Sink with the nozzles aligned with the holes in the Silicone Mat (do not use excessive pressure). 6. Place the Elution Riser block into the right hand side of the vacuum chamber and position the Elution Plate on top of the Riser. 7. On the platform layout, the reagents required, the positions of the reagent tubs and the volume of the reagents required will be highlighted in pink (areas R1 and C1). a. If less than 96 samples in 12 columns are to be extracted, then edit the programme to set the appropriate number of columns. i. Open the wizard and press [Clear All] ii. Click on the required columns, click [Jump to End], then click [Finish]. b. Fill the reagent tubs with the solutions displayed and the required volumes shown on the screen. The tubs have graduated measurements on the side for ease of volume measurement. Always ensure slightly more reagent (~5ml) is added to each tub to avoid running out during the run. The Lysis Buffer is already made up in excess. a. The reagent tubs must have their lids in place before the run starts b. Reagent tubs are reusable. Label the tubs, and rinse with water before each use. 8. The Tip Racks are placed on positions B2 and C2 using the mouse and highlight the tips that are available for use. 9. Check that the lids are removed the Tip Racks and Elution Plate, check that the cover has been removed from the waste-tip shute and the waste tip collector is in place. 10. Ensure the elution volume is set to 80ul. 11. Click on [Start] then tick the checklist and click [OK] to begin the run. 6

7 a. The QIAxtractor pauses after the vacuum step has drawn the sample through the Capture Plate. Check that the wells are not blocked. If clear, click on [Continue]. b. Blocked well can be cleared by puncturing the membrane with a needle. Use a clean needle for each blocked membrane, record the plate positions and then click on [Continue]. 12. When the run has completed, place the lid on the Elution Plate (containing extracted RNA), remove from the robot and place on ice. Follow RT protocol as described (see section 3). The RT and subsequent PCRs can be performed in 96 well plates. 13. After the run has completed, decontamination needs to be performed before the start of the next run. a. Remove the Capture Plate, Silicone Mat and empty tip racks and discard. b. Relid unsued tips and remove from the platform. c. Remove reagent tubs, discard any remaining liquid to waste, wash in tap water, rinse in distilled water and leave to dry. d. Remove the Waste Sink and Elution Riser, wash in tap water, rinse in distilled water and leave to dry. DO NOT heat or autoclave any of the washed components. e. Remove the Transfer Carriage, wipe the Transfer Carriage and the platform with nucleic acid decontamination wipes followed by alcohol wipes. f. Add 50ml of water to the drain and purge by clicking on the bucket icon and follow the instructions. g. Dry the vacuum chamber with paper towel, wipe with nucleic acid decontamination wipes followed by alcohol wipes. h. Cover the tip shute with the metal cover provided. i. Run the UV decontamination cycle by pressing the bulb icon and follow the instructions. j. When the UV cycle is complete, replace all the washed plastic reagent tubs, the Elution Rise, Transfer Carriage and Waste Sink back on the 7

8 platform, ready for the next run and repeat the UV decontamination cycle. k. When the UV cycle is complete, remove the tip shute cover, switch off the robot and then shut-down the computer. 8

9 3. Random Priming RT 1. Prepare RT mix for N + 2 (N = No. of tubes in test) 2. 1x mix 1. 10X buffer II (Invitrogen 4 ) 7.0ul 2. 50mM MgCl 2 7.0ul 3. Random primers 5 4. dntps (10mM) 2.0ul 5. M-MLV (200U/ul) Invitrogen 2.0ul 6. RNase-free H 2 O ul 3. Transfer 40ul of extracted nucleic acid to a PCR tube. 4. Denature the dsrna at 97 o C for 5 min. 5. Chill the tubes on ice for 2 min. 6. Add 30ul to each tube containing the extracted RNA. 7. Incubate the tubes at 37 o C for 1h. 8. Incubate the tubes at 95 o C for 5 min. 9. Chill the tubes on ice for 2 min. 10. The total volume should be 70ul. 11. The cdna can be used directly in the PCR, and stored at -20 o C for further use. 4 RT and PCR conditions have been optimised using Invitrogen reagents. Re-optimisation may be required if other reagents are used 5 See section 9 9

10 4. Rotavirus VP6-specific PCR 6 1. Prepare PCR mix for N + 2 (N = No. of tubes in test) 1x mix 10X buffer II (Invitrogen) 50mM MgCl2 dntps (10mM) Taq Polymerase (5U/ul) (Invitrogen) primer VP6-F 7 (20pmoles/ul) primer VP6-R (20pmoles/ul) RNase-free H2O 4.5ul 2.0ul 0.2ul 35.3ul 45.0ul 2. Add 45ul of PCR mix to each PCR tube. 3. Add 5ul cdna (from the RT reaction: section 3). 4. Briefly spin in microcentrifuge (pulse for 5 sec). 5. Transfer tubes to the PCR machine. 6. Add tubes to thermocycler and cycle at the following temperatures for the following times: 94 o C 2min X1 94 o C 1min 55 o C 1min X35 72 o C 1 min 72 o C 7min X1 15 o C Hold 6 This PCR is designed for detecting rotavirus RNA in clinical samples, and can be used for confirmation of rotavirus ELISA-equivocal results and SG determination when followed by sequencing. 7 See section 8 for primer sequences 10

11 5. Rotavirus VP6-specific real-time PCR (Taqman) 1. Prepare PCR mix for N + 2 (N = No. of tubes in test) 1x mix Taqman MasterMix 2x (Invitrogen) ROX (Invitrogen) primer VP6-F 8 (20pmoles/ul) primer VP6-R (20pmoles/ul) VP6probe (20uM) RNase-free H2O 12.5ul 0.5ul 0.2ul 7.8 ul 23.0ul 2. Add 23ul of Taqman mix to each well of a 96 well plate. 3. Add 2ul cdna (from the RT reaction: section 3). 4. Ensure there are no bubbles in the wells and seal the plate with the optical covers. 5. Briefly spin in plate centrifuge (pulse for 5 sec). 6. Transfer plate to and ABI PRISM 7500 SDS Taqman machine. 7. Cycle at the following temperatures for the following times: 50 o C 2min X1 95 o C 2min X1 95 o C 15sec 60 o C 1min X35 All the wells should be assayed using the dye and quencher of the probe 8 See section 8 for primer sequences 11

12 6. Rotavirus NSP4-specific PCR 9 1. Prepare PCR mix for N + 2 (N = No. of tubes in test) 1x mix 10X buffer II (Invitrogen) 50mM MgCl2 dntps (10mM) Taq Polymerase (5U/ul) (Invitrogen) primer NSP4-F 10 (20pmoles/ul) primer NSP4-R (20pmoles/ul) RNase-free H2O 4.5ul 2.0ul 0.2ul 35.3ul 45.0ul 2. Add 45ul of PCR mix to each PCR tube 3. Add 5ul cdna (from the RT reaction: section 3) 4. Briefly spin in microcentrifuge (pulse for 5sec). 5. Transfer tubes to the PCR machine. 6. Add tubes to thermocycler and cycle at the following temperatures for the following times: 94 o C 2min X1 94 o C 1min 48 o C 1min X35 72 o C 1 min 72 o C 7min X1 15 o C Hold 9 This PCR is designed for NSP4-genotype characterisation when followed by sequencing. 10 See section 8 for primer sequences 12

13 7. Rotavirus genotyping PCRs 7.1. G-Typing consensus PCR (VP7) 1. Prepare 1 st round PCR mix for N + 2 (N = No. of tubes in test) 1x mix 10X buffer II (Invitrogen) 50mM MgCl2 dntps (10mM) Taq Polymerase (5U/ul) (Invitrogen) primer VP7-F 11 (20pmoles/ul) primer VP7-R (20pmoles/ul) RNase-free H2O 2. Add 45ul of PCR mix to each PCR tube. 3. Add 5ul cdna (from the RT reaction). 4. Briefly spin in microcentrifuge (pulse for 5sec). 5. Transfer tubes to the PCR machine. 4.5ul 2.0ul 0.2ul 35.3ul 45.0ul 6. Add tubes to thermocycler and cycle at the following temperatures for the following times: 94 o C 2min X1 94 o C 1min 52 o C 1min X35 72 o C 1min 72 o C 7min X1 15 o C Hold 11 These primers are preferred to those of Gouvea et al, as they provide increased sensitivity and sufficient DNA for direct sequencing if the genotype in not determined in the 2 nd round multiplex reaction. See section 8 for primer sequences 13

14 7.2. G-Typing multiplex PCR 1. Prepare 2 nd round PCR mix for N + 2 (N = No. of tubes in test) 1x mix 10X buffer II (Invitrogen) 4.8ul 50mM MgCl2 2.5ul dntps (10mM) Taq Polymerase (5U/ul)(Invitrogen) 0.2ul primer VP7-R 12 (20pmoles/ul) primer G1 (20pmoles/ul) primer G2 (20pmoles/ul) primer G3 13 (20pmoles/ul) primer G4 (20pmoles/ul) primer G8 15 (20pmoles/ul) primer G9 14 (20pmoles/ul) primer G10 15 (20pmoles/ul) primer G12 16,15 (20pmoles/ul) RNase-free H2O 30.5ul 1. Add 48ul round 2 mix to each PCR tube. 2. Add 2 ul of first round product (see section 6.1). 3. Briefly spin in microcentrifuge (pulse for 5sec). 4. Transfer tubes to the PCR machine. 5. Add tubes to thermocycler and cycle using the following programme: 94 o C 4min X1 94 o C 1min 42 o C 2min X30 72 o C 1 min 72 o C 7min X1 15 o C Hold 12 See section 8 for primer sequences 13 The position and sequence of the G3 primer is different from that previously published by Gouvea et al. These changes have been introduced in order to prevent cross-reactivity between G3 and G10 primers and targets. 14 The G9 primer has been modified to take account of the genetic drift of G9 strains from the prototype strain. 15 A G10 primer has been included as increased numbers of G10 strains have been isolated in India in recent years. 16 A G12 primer has been included as increased numbers of G12 strains have been isolated in India in recent years. 15 The G8 and G12 primers have been modified to overcome mistyping 14

15 7.3. VP7 semi- nested consensus PCR (for sequence typing) Prepare PCR mix for N + 2 (N = No. of tubes in test) 1x mix 10X buffer II (Invitrogen) 50mM MgCl2 dntps (10mM) Taq Polymerase (5U/ul) (Invitrogen) primer VP7-F 18 (20pmoles/ul) primer VP7-RINT (40pmoles/ul) 19 RNase-free H2O 2. Add 45ul of PCR mix to each tube. 3. Add 2ul 1 st Round amplicon from (see section 6.1). 4. Briefly spin in microcentrifuge (pulse for 5sec). 5. Transfer tubes to the PCR machine. 4.5ul 2.0ul 0.2ul 35.3ul 45.0ul 6. Add tubes to thermocycler and cycle at the following temperatures for the following times: 94 o C 2min X1 94 o C 1min 50 o C 1min X35 72 o C 1min 72 o C 7min X1 15 o C Hold 17 This PCR provides increased sensitivity and should be used in conjunction with DNA sequencing for samples which fail to G-type using the method described in section See section 8 for primer sequences pmoles of primer should be used to take account of its degenerate nature. 15

16 7.4 P-Typing consensus PCR (VP4) 1. Prepare 1 st round PCR mix for N + 2 (N = No. of tubes in test) 1x mix 2. Add 45ul to each tube. 3. Add 5ul cdna. 10X buffer II (Invitrogen) 50mM MgCl2 dntps (10mM) Taq Polymerase (5U/ul) (Invitrogen) Primer VP4F 20 (20pmoles/ul) Primer VP4R (20pmoles/ul) RNase-free H2O 4. Briefly spin in microcentrifuge (pulse for 5sec). 5. Transfer tubes to the PCR machine. 4.5ul 2.5ul 0.2ul 34.8ul 45.0ul 6. Add tubes to thermocycler and cycle at the following temperatures for the following times : 94 o C 2min X1 94 o C 1min 50 o C 1min X35 72 o C 1 min 72 o C 7min X1 15 o C Hold 20 See section 8 for primer sequences 16

17 7.5. P-Typing multiplex PCR 1. Prepare 2nd round mix for N + 2 (N = No. of tubes in test) 1x mix 10X buffer II (Invitrogen) 4.8ul 50mM MgCl2 2.5ul dntps (10mM) Taq Polymerase (5U/ul) (Invitrogen) 0.2ul primer VP4F 21 (20pmoles/ul) primer P[4] (20pmoles/ul) primer P[6] (20pmoles/ul) primer P[8] 22 (20pmoles/ul) primer P[9] (20pmoles/ul) primer P[10] (20pmoles/ul) primer P[11] 23 (20pmoles/ul) RNase-free H2O 32.5ul 48.0ul 2. Add 48ul 2 nd round mix to a new 0.2ml tube 3. Add 2ul of first round product (see section 6.4). 4. Briefly spin in microcentrifuge (5sec). 5. Transfer tubes to PCR machine room. 6. Add tubes to thermocycler and cycle at the following temperatures for the following times: 94 o C 4min X1 94 o C 1min 45 o C 2min X30 72 o C 1 min 72 o C 7min X1 15 o C Hold 21 See section 8 for primer sequences 22 The P[8] primer has been modified to take account of the genetic drift of P[8] strains from the prototype strain. 23 A P[11] primer has been included as increased numbers of P[11] strains have been isolated in India in recent years. 17

18 8. Primer sequences VP6 oligonucleotide primers (Iturriza Gómara et al 2002, J Virol) VP6-F 5 GAC GGV GCR ACT ACA TGG T 3 VP6-R 5 GTC CAA TTC ATN CCT GGT G 3 Product: 382bp VP6probe for real-time PCR (Taqman) (Iturriza et al unpublished) VP6probe FAM 5 CCA CCR AAY ATG ACR CCA GCN GTA 3 MGB NSP4 oligonucleotide primers (Ciarlet et al 2000, Virology) NSP4-F 5 GGC TTT TAA AAG TTC TGT TCC GAG 3 NSP4-R 5 GGT CAC ACT AAG ACC ATT CC 3 Product size: 743bp V= A or C or G Y= C or T R= A or G N= A or T or C or G W=A or T 18

19 G-typing oligonucleotide primers 1. First round consensus primers (Iturriza Gómara et al, 2001, J Clin Microbiol) VP7-F 5' ATG TAT GGT ATT GAA TAT ACC AC 3' (nt 51-71) VP7-R 5' AAC TTG CCA CCA TTT TTT CC 3' (nt ) Product size: 881bp 2. Second round typing primers: All serotyping primers are included in the mix [Gouvea et al., 1990, J Clin Microbiol; Iturriza Gómara et al, 2004, J Clin Microbiol; Aladin et al., unpublished] Genotype G1: G1 5' CAA GTA CTC AAA TCA ATG ATG G 3' (nt ) product size; 618bp Genotype G2: G2 5' CAA TGA TAT TAA CAC ATT TTC TGT G 3' (nt ) product size; 521bp Genotype G3: G3 5' ACG AAC TCA ACA CGA GAG G 3' (nt ) product size; 682bp Genotype G4: G4 5' CGT TTC TGG TGA GGA GTT G 3' (nt ) product Size; 452bp Genotype G8: G8 5' TTR TCG CAC CAT TTG TGA AAT 3' (nt ) product size; 756bp Genotype G9: G9 5 CTT GAT GTG ACT AYA AAT AC 3 (nt ) product size; 179bp Genotype G10: G10 5 ATG TCA GAC TAC ARA TAC TGG 3 (nt ) product size; 266bp Genotype G12 G12 5 GGT TAT GTA ATC CGA TGG ACG 3 (nt ) product size; 396bp 3. VP7 nested consensus primer (for sequence typing) VP7-RINT 5 ANA YNG ANC CWG TYG GCC A 3 (nt ) Product size; 293bp Y= C or T W= A or T R= A or G 19

20 P-typing oligonucleotide primers 1. First round consensus primers [Gentsch et al., 1992, J Clin Microbiol] VP4F 5' TAT GCT CCA GTN AAT TGG 3' (nt ) VP4R 5' ATT GCA TTT CTT TCC ATA ATG 3' (nt ) Product size: 663bp 2. Second round typing primers: All serotyping primers are included in the mix [Gentsch et al., 1992, J Clin Microbiol;Iturriza-Gómara et al., 2000, J Virol; 2004, J Clin Microbiol] P[4] (previously genogroup 2) 2T-1 5' CTA TTG TTA GAG GTT AGA GTC 3' (nt ) product size; 483bp P[6] (previously genogroup 3) 3T-1 5' TGT TGA TTA GTT GGA TTC AA 3' (nt ) product size; 267bp P[8] (previously genogroup 1) 1T-1D 5 TCT ACT GGR TTR ACN TGC 3 (nt ) product size; 345bp P[9] (previously genogroup 4) 4T-1 5' TGA GAC ATG CAA TTG GAC 3' (nt ) product size; 391bp P[10] (previously genogroup 5) 5T-1 5' ATC ATA GTT AGT AGT CGG 3' (nt ) product size; 583bp P[11] P[11] 5' GTA AAC ATC CAG AAT GTG 3' (nt ) product size; 312bp N=A or T or G or C R= A or G 20

21 Diagrammatic illustration of VP7 and VP4 specific primer positions and type-specific amplicon sizes. Group A rotavirus gene 9 (VP7) NA ' 3' Amplification products 881bp 756b p first round copy of gene 9 Genotype G8 Genotype G3 682b p Genotype G1 618b p Genotype G2 primer s 521b p 452b p 396bp Genotype G4 Genotype G12 Genotype G10 Group A rotavirus gene 4 (VP4) 266b p 179b p Genotype G9 NT ' 3' Amplification products 663bp First Round Amplicon 146bp P[6] 191bp 224bp P[11] P[8] 270bp P[9] 362bp P [4] 462bp P[10] 21

22 9 RNA extraction reagents a. Preparation of solutions 1M Tris Dissolve 121.1g of Tris base in 800ml of H 2 O Adjust the ph to the desired value by adding concentrated HCl ph HCl 7.4 ~70ml 7.6 ~60ml 8.0 ~42ml 0.5M EDTA ph 8.0 Add 186.1g of disodium EDTA to 800ml H 2 O Adjust the ph to 8.0 with NaOH (~ 20g NaOH pellets) L6 Buffer L2 Buffer guanidinium isothiocyanate (GTC) 60g 0.1M Tris-HCl ph ml 0.2M EDTA ph ml Triton X g 70% ethanol GTC 180g 0.1M Tris-HCl ph ml Ethanol cell culture grade distilled H 2 O 70ml 30ml b. Preparation of size fractionated silica 1. Add 60g silicon dioxide, SiO 2 (Sigma; S-5631) to demineralised water to a total volume of 500ml in a measuring glass cylinder. 2. Allow the silica to sediment under gravity for 24h at room temperature 3. Extract 430ml of supernatant and add demineralised water to 500ml and shake vigorously. 4. Sediment for 24h at room temperature. 5. Extract 440ml supernatant and add 600ul HCl (32%, w/v) to adjust the silica suspension to ph

23 Note: Wear visor, apron and gloves when handling concentrated acids. 6. Aliquot the silica suspension in 4ml volumes in glass bijoux bottles and sterilise by autoclaving. c. Preparation of RNase-free deionised water 1. Add 100ul of diethylpyrocarbonate (DEPC)(Sigma: D-5758) to 100ml of cell culture grade deionised water. NOTE: Perform the addition in a fume cupboard. 2. Incubate for >12hr at 37 o C. 3. Autoclave for 15min. d. Random primers (Invitrogen: ) e. TE buffer Add 450ul of RNase-free water to aliquot, use 1ul/50ul reaction volume. Tris-HCl, ph 7.4 EDTA, ph mM 1mM f. Sample buffer (gel loading buffer) Ficoll 10% Orange G 0.25% in TE buffer g. Ethidium bromide (EtBr) 10mg/ml in TE buffer (if adding to the gel, 20ulEtBr solution in 50ml agarose gel) 23

24 10. Disposal of toxic waste Waste should be discarded in accordance with local guidelines. The following identifies toxic substances used in the procedures described. Ethidium bromide 1. Ethidium bromide staining solution should be collected and stored for disposal 2. Ethidium bromide stained gels should be discarded in a labelled discard jar and stored for disposal Guanidinium isothiocyanate Add GTC to an equal volume of 10M NaOH and store for disposal NOTE: GTC will release cyanate gas under acidic conditions Solvents Collect ethanol and acetone in a labelled Winchester bottle and store for disposal 24

25 11. Agarose- gel Electrophoresis 1. Add 2g of UltraPure Agarose 100 (Invotrogne Ct No ) (or an appropriate gel for the analysis of nucleic acid amplicons of <1Kb) to 100ml 1X TBE for a midi gel to give a 2% gel. 2. Melt in microwave at full power for 2 minutes. Microwave procedure Caution: Any microwaved solution may become superheated and boil vigorously when moved or touched. Use extreme care in handling. Remove the boiling solution from the microwave oven, allow to stand for a few seconds at room temperature and release the air by gentle swirling. Heat, using several short, 20 to 30 second intervals with gentle swirling between pulses to resuspend the powder. 4. Cool to 45 o C then pour into gel plate (size 14 cm [W] x 16 cm [L]) 24 fitted with two slot combs (to give a well of 20ul capacity). 5. Add 10ul of PCR product to 10ul sample buffer in a microtitre plate. 6. Remove the comb and end pieces and add 20ul of size markers 25 or diluted sample to the appropriate well. 6. Place the gel plate in the gel tank and add 1X TBE level with the gel (do not flood the plate at this stage) 7. Run the products into the gel for 5min at a constant voltage of 150V. 8. Flood the gel with 1X TBE making sure it is fully submerged 9. Electrophorese at constant Voltage (between 5 and 8 V/cm). 24 Avoid the use of mini gels for genotyping 2 nd round PCRs as the resolution may not be sufficient to differentiate genotypes. 25 Use a 100 bp ladder for DNA fragments of between 100 and 1000 bp. 25