Kleargene plant spin plate DNA extraction kits user manual
Contents
1. Table 4 Reagents to be added to buffers from 384 well plate kits For any queries on this manual or running KASP reactions in your laboratory please contact The volumes of each reagent to add are also declared on the labels of the bottles in the 4 x 16 x 64 x 80 x and 400 x kits space constraints prevent this on the bottles of the 1 x kit All locations except USA Email tech support lgcgroup com Phone 44 0 1992 476 486 USA only Email us support lgcgroup com Phone 1 978 338 5317 Buffers L1 amp C1 These buffers require the addition of 100 B mercaptoethanol before use It is recommended that any buffer to which B mercaptoethanol is added should be used immediately L1 and C1 should be aliquoted appropriately if not to be used in its entirety The enzyme a amylase may also be added to L1 if seeds are being extracted see the Notes section at the end of this document for information regarding its use If required it is recommended that a amylase is added to L1 to a final concentration of 300U mL Buffers A1 amp W1 Buffer A1 requires the addition of ethanol and isopropanol Buffer wie requires the addition of ethanol only It is not necessary to aliquot these buffers before use It is however recommended that any unused buffer containing these alcohols should be stored out of direct sunlight and that the bottle caps are securely tightened to ensure that no evaporation occurs 4 Safet2. the protocol was developed and tested on the following sources Barley Hordeum vulgare e Sweet pepper Capsicum annuum Cowpea Vigna unguiculata leaves and seeds Lettuce Lactuca sativa e Tomato Solanum lycopersicum leaves and seeds Maize Zea mays leaves and kernels Soy Glycine max L e Wheat Triticum aestivum leaves and seeds Sugar beet Beta vulgaris DNA is not significantly fragmented by the method with fragment sizes of gt 50 kb typically seen The absence of nucleases in samples prepared with Kleargene has been demonstrated by overnight incubation at 37 C in the presence of 10 mM MgCl For any queries on this manual or running KASP reactions in your laboratory please contact All locations except USA Email tech support lgcgroup com Phone 44 0 1992 476 486 USA only Email us support lgcgroup com Phone 1 978 338 5317 6 1 6 3 For any queries on this manual or running KASP reactions in your laboratory please contact Leaf extractions The protocol assumes you have lyophilised or dessicated your samples before extraction this allows you to homogenise the samples dry before extraction which is most efficient and allows you to extract from relatively more sample than would otherwise be the case up to 30 mg Drying samples in an oven is not recommended as the heat degrades the sample When processing very small amounts of sample e g 1 or 2 punches it is possible to homog
3. Kleargene plant spin plate DNA extraction kits user manual Contents of this guide a A O N Product description and specification Kit contents Reagent preparation Safety information Protocol 5 1 Cell lysis 5 2 Absorption of DNA to glass fibre membranes 5 3 Contaminant removal through washing 5 4 Elution of purified DNA General points 6 1 Leaf extractions 6 2 Seed extractions 6 3 Adapting the protocol to your laboratory Troubleshooting Frequently asked questions 1 Product description and specification The Kleargene family are a series of kits designed to enable simple rapid extraction and purification of genomic DNA from a variety of sources The Kleargene plant plate DNA kit is designed for the extraction of DNA from small amounts 20 30 mgs of plant material The method is based on the highly proven technology of detergent driven cell lysis followed by guanidinium isothiocyanate mediated DNA binding to glass fibre membranes encapsulated in a filter plate Contaminants are removed by washing and DNA is subsequently eluted into a low salt buffer The entire process is carried out in one well per sample and full 96 well or 384 well plates of samples can be extracted in under an hour 2 Kit contents Component Storage KBS 1012 201 KBS 1012 202 KBS 1012 210 KBS 1012 211 Buffer L1 20 25 C 15 mL 60 mL 250 mL 1000 mL Buffer C1 20 25 C 30 mL 125 mL 500 mL 2 x 1000 mL B
4. call 1 978 338 5317 www lgcgroup com genomics genomicsQlgcgroup com Science for a safer world Brazil e Bulgaria China Czech Republic Finland France Germany Hungary India Ireland Italy e Netherlands Poland Romania Russia South Africa Spain Sweden Turkey United Kingdom USA All trademarks and registered trademarks mentioned herein are the property of their respective owners All other trademarks and registered trademarks are the property of LGC and its subsidiaries Specifications terms and pricing are subject to change Not all products are available in all countries Please consult your local sales representative for details No part of this publication may be reproduced or transmitted in any form or by any means electronic or mechanical including photocopying recording or any retrieval system without the written permission of the copyright holder O LGC Limited 2014 All rights reserved 4046 CF 0714
5. d if it is available e 384 well filter plate kits There is insufficient capacity in the wells of the filter plate to allow the entire mixture to be transferred in one go 100 uL of the mixture should be transferred the rest of the mixture can be extracted using the same filter plate at a later date if required see the Notes section for details regarding this 9 Incubate the filter plates for 2 minutes at room temperature then centrifuge the filter plate on top of the deep well collection plate or reservoir at 3 000 x g for 2 minutes 10 Clear spin through from the collection plate reservoir and place the filter plate back on top of it 5 3 Contaminant removal through washing 11 Add buffer A1 to each sample well on the filter plate e 96 well filter plate kits Add 150 uL e 384 well filter plate kits Add 75 ul 12 Incubate the filter plates for 2 minutes at room temperature then centrifuge the filter plate on top of the deep well collection plate or reservoir at 3 000 x g for 2 minutes 13 Clear spin through from the collection plate reservoir and place the filter plate back on top of it 14 Add buffer W1 to each sample well on the filter plate e 96 well filter plate kits Add 75 uL e 384 well filter plate kits Add 37 5 uL 15 Incubate the filter plates for 2 minutes at room temperature then centrifuge the filter plate on top of the deep well collection plate or reservoir at 3 000 x g for 2 minutes 16 Clear s
6. enise the sample in buffer L1 rather than beforehand this allows you to handle samples without prior drying Note that if the homogenisation is carried out in buffer L16 then overloading of sample will lead to inefficient disruption and lower yields downstream Younger and fresher source leaves give the best results Seedlings can be used although monocots may prove tougher to disrupt fully Etiolated samples are not generally required the compositions of the buffers used are sufficient to deal with the metabolites normally found in leaf material Seed extractions Extracting DNA from seeds presents a slightly different challenge to extracting DNA from leaves the concentration of potentially interfering metabolites is much lower but the presence of starch can cause blockages to the membranes of filter plates e Seeds are highly variable in size and in the case of smaller seeds e g tomato pepper careful aspiration of the L10 c1 lysate mixture will probably be OK as it should be possible to avoid transferring significant amounts of starch over to the filter plate this way e For bigger seeds e g com kernels the amounts of starch produced after grinding will cause significant blocking issues downstream in these instances it is recommended that the enzyme a amylase is added to buffer L1 The a amylase degrades insoluble starch into a mixture of dextrins that are soluble in L1 thus allowing the cleared lysate to pass through
7. is required This will inevitably reduce the overall concentration of the eluted DNA e You are supplied with an approximate two fold excess of each buffer over the volumes needed to carry out an extraction exactly as defined in the protocol This allows you flexibility at certain stages of the protocol The ratios of buffers used must be maintained 1 volume L1 2 volumes C1 2 volumes A1 1 volume W1 1 volume EtOH 1 volume E1 e Many plant materials occlude a certain proportion of buffer L16 such that not all 75 uL is available for downstream processing It is acceptable to add additional L1 initially However if this is done you must also increase the volumes of all other buffers in proportion The protocol is likely to fail if you do not do this For any queries on this manual or running KASP reactions in your laboratory please contact All locations except USA Email tech support lgcgroup com Phone 44 0 1992 476 486 USA only Email us support lgcgroup com Phone 1 978 338 5317 7 Troubleshooting Problem Likely cause Explanation suggestions Excess starch in lysate Blocked filter Add a amylase to buffer L1 before use seeds only plate Excessive starting material Use less starting material in future extractions Insufficient disruption of tissue Ensure that tissue samples are ground prior to addition of buffer L1 homogenised before use Ensure that the amount of tissue used is withi
8. n the recommended range up to 30 mg dry tissue Low DNA yield Insufficient lysis Excess starting material will reduce the ability of Buffer E1 not pre warmed to 55 C before use cells to lyse Insufficient starting material will result in lower than expected yields DNA elution from the filter plate is much more efficient when buffer E1 is at 55 C Poor sample storage prior to extraction Ensure that tissues are stored appropriately to minimise DNA degradation It is recommended that all plant tissue should be stored at 20 C or lower Younger and fresher source tissue samples may produce better quality DNA DNA is degraded Tissue samples were dried in an oven Oven drying of samples is not recommended as heat degrades plant tissue Lyophilise tissue for storage prior to performing DNA extraction or grind up fresh tissue in liquid nitrogen and store at 80 C Precipitate has formed in buffer L1 lysis buffer Incubate buffer at 37 C and mix well until clear Ethanol carryover Ensure that the spin plate is completely dry after the final wash step with ethanol DNA does not Salt carryover perform well in y Ensure that the wash buffers A1 and w1 are at room temperature before use downstream experiments Insufficient excessive DNA used in downstream experiments Optimise the quantity of DNA that is used in downstream experiments with a DNA dilution series Too much
9. or too little DNA can adversely affect experimental performance For any queries on this manual or running KASP reactions in your laboratory please contact 10 All locations except USA Email tech support lgcgroup com Phone 44 0 1992 476 486 USA only Email us support lgcgroup com Phone 1 978 338 5317 8 Frequently asked questions 1 What type of centrifuge is required for the Kleargene plant tissue protocol The centrifuge will need an adapter suitable for plates and to be capable of achieving 3000 x g 2 Can the Kleargene buffers be purchased individually Yes All buffers can be purchased individually Buffer Volume Product code Li 250 mL KBS 1012 306 c19Q 500 mL KBS 1012 308 me 250 mL KBS 1012 309 wi 75 mL KBS 1012 310 E10 250 mL KBS 1012 311 Additional filter plates can also be purchased Plate size Pack size Product code 96 well 10 plates KBS 1012 303 384 well 10 plates KBS 1012 304 3 Can the Kleargene kit be used for the extraction of RNA from plant tissue No If you are working with large numbers of samples why not consider our Genespin platform The Genespin enables semi automated high throughput DNA extractions from plant tissues and utilises Kleargene chemistry For any queries about this guide please contact All locations except USA email tech support lgcgroup com or call 44 0 1992 476 486 USA only email us support lgcgroup com or
10. pin through from the collection plate reservoir and place the filter plate back on top of it 17 Add 100 ethanol to each sample well on the filter plate 96 well filter plate kits Add 75 uL e 384 well filter plate kits Add 37 5 uL For any queries on this manual or running KASP reactions in your laboratory please contact 6 All locations except USA Email tech support lgcgroup com Phone 44 0 1992 476 486 USA only Email us support lgcgroup com Phone 1 978 338 5317 18 19 Incubate the filter plates for 2 minutes at room temperature then centrifuge the filter plate on top of the deep well collection plate or reservoir at 3 000 x g for 2 minutes Incubate the filter plates on their own for 10 minutes at 50 70 C to eliminate any residual ethanol 5 4 Elution of purified DNA 20 21 22 6 Replace the deep well collection plate reservoir used up to this point with an elution plate and place the filter plate back on top of it Add buffer E1 pre warmed to 55 C to each sample well on the filter plate N B Yield is significantly reduced if the elution buffer is not pre warmed to 55 C e 96 well filter plate kits Add 75 uL e 384 well filter plate kits Add 37 5 uL Incubate at 55 C for 5 minutes then centrifuge the filter plate on top of the deep well collection plate at 3 000 x g for 2 minutes to elute the DNA General points The protocol is applicable in principle to any plant however
11. the filter plate membrane Adapting the protocol to your laboratory If necessary the extraction process can be suspended after any washing step and the plates can be kept at room temperature for many hours If the protocol is halted for an extended period of time at any point before step 7 the lysate mixtures should be refrigerated to limit the oxidation of the plant compounds that have been released into solution e If this is done it is vital that the mixtures are thoroughly warmed and mixed well before continuing the process as certain buffer components are liable to form precipitates All locations except USA Email tech support lgcgroup com Phone 44 0 1992 476 486 USA only Email us support lgcgroup com Phone 1 978 338 5317 e When using the 384 well filter plates the well capacity of the filter plates limits the volume of sample that can be processed at one time e The protocol above specifies how 100 uL of the L1 C16 lysate mixture should be extracted If additional yield is required it is possible to repeat the extraction process using the same filter plate after the initial elution has been carried out be careful to ensure the correct orientation of the filter plate and samples if this is done e A single elution typically yields 70 80 of the total DNA bound to the glass fibre in the filter plates a second elution step can be carried out to remove the majority of all the remaining DNA if maximal yield
12. ue homogenisation instruments and consumables Manual pipettes and disposable pipette tips OR a microplate dispensing robot Polypropylene storage plates and optionally reservoir plates Fan oven Waterbath incubator Personal protective equipment lab coat gloves safety glasses 3 Reagent preparation Precipitates can form in the lysis and binding buffers after prolonged low temperature storage incubate at 37 C if this is the case and mix well until clear Ensure that the buffers are well mixed before use following the addition of the necessary reagents see Tables 3 and 4 below Component Reagent to add 1x96 4x 96 16 x 96 64 x 96 Buffer L1 amp B mercaptoethanol 120 uL 480 uL 2 mL 8 mL Buffer C1 B mercaptoethanol 240 uL 1 mL 4 mL 2x8 mL Isopropanol 7 5 mL 30 mL 125 mL 2 x 250 mL Buffer A1 Ethanol 7 5 mL 30 mL 125 mL 2 x 250 mL Buffer W1 Ethanol 10 5 mL 42 mL 175 mL 700 mL Table 3 Reagents to be added to buffers from 96 well plate kits Component Reagent to add 1 x 384 4x 384 16 x 384 80 x 384 400 x 384 Buffer L1 B mercaptoethanol 480 uL 2mL 8 mL 40mL 200 mL Buffer C1 B mercaptoethanol 1 mL 4 mL 2x8 mL 2 x 40mL 2 x 200 mL Isopropanol 30 mL 125 mL 2x 250 mL 2x1250mL 2 x 6250 mL Buffer Aj eea a imam OSE a NES ieee Ethanol 30 mL 125 mL 2x 250 mL 2x 1250mL 2 x 6250 mL Buffer W1 Ethanol 42 mL 175 mL 700 mL 3500 mL 17 500 mL
13. uffer A1 20 25 C 15 mL 60 mL 250 mL 2 x 500 mL Buffer W1 20 25 C 4 5 mL 18 mL 75 mL 300 mL Buffer E1 20 25 C 15 mL 60 mL 250 mL 1000 mL Filter plate 20 25 C 1x96 4x96 16 x 96 64 x 96 Table 1 Contents of Kleargene 96 well plant kits Component Storage KBS 1012 204 KBS 1012 205 KBS 1012 212 KBS 1012 213 KBS 1012 214 Buffer L1 20 25 C 60 mL 250 mL 1000 mL 5000 mL 25000 mL Buffer C1 20 25 C 125 mL 500 mL 2x1000mL 2x5000 mL 2x 25000 mL Buffer A1 20 25 C 60 mL 250 mL 2 x 500 mL 2x 2500 mL 2x 12500 mL Buffer W168 20 25 C 18 mL 75 mL 300 mL 1500 mL 7500 mL Buffer E1 20 25 C 60 mL 250 mL 1000 mL 5000 mL 25000 mL Filter plate 20 25 C 1x 384 4 x 384 16 x 384 80 x 384 400 x 384 Table 2 Contents of Kleargene 384 well plant kits 7 before the required additional reagents are added to each buffer 2 this should be stored in the dark To be supplied by the user Additional reagents Ethanol Isopropanol propan 2 ol B mercaptoethanol 2 mercaptoethanol a amylase ONLY for seed material Sigma Aldrich cat no A3403 recommended For any queries on this manual or running KASP reactions in your laboratory please contact 2 All locations except USA Email tech support lgcgroup com Phone 44 0 1992 476 486 USA only Email us support lgcgroup com Phone 1 978 338 5317 Equipment needed Tiss
14. with the second step Add 75 uL buffer L1 to the ground sample and mix well Incubate the samples e If processing leaf material or seed material without the addition of a amylase to L1 incubate at 55 C for 10 minutes e If processing seed material with the addition of a amylase to L1 incubate at 91 C for 30 minutes in a waterbath Add 150 uL buffer C1 to each lysed sample and mix until the solution is homogenous Incubate samples at 55 C for 10 minutes Centrifuge your samples at 3 000 x g for 2 minutes to clear the mixtures of most cellular debris Absorption of DNA to glass fibre membranes Place the filter plate on top of another deep well plate or reservoir e 96 well filter plate kits Use a plate with a capacity of at least 300 uL well or an equivalent reservoir e 384 well filter plate kits Use a plate with a capacity of at least 200 UL well or an equivalent reservoir For any queries on this manual or running KASP reactions in your laboratory please contact All locations except USA Email tech support lgcgroup com Phone 44 0 1992 476 486 USA only Email us support lgcgroup com Phone 1 978 338 5317 Transfer the cleared lysate binding buffer mixture into the wells of the filter plate ensuring that as little debris as possible is transferred along with the cleared lysate excess debris may block the membranes in the filter e 96 well filter plate kits The entire mixture can be transferre
15. y information DO NOT ADD BLEACH OR ACIDIC SOLUTIONS DIRECTLY TO THE SAMPLE PREPARATION WASTE e The sample preparation waste contains guanidinium isothiocyanate which can form highly reactive compounds when combined with bleach If liquid containing these buffers is spilt clean with suitable laboratory detergent and water If spilt liquid contains potentially infectious agents clean the affected area first with laboratory detergent and water and then with 1 v v sodium hypochlorite e It is highly recommended that personal protective equipment is worn throughout the extraction process For more detailed information please refer to the safety data sheets SDS For any queries on this manual or running KASP reactions in your laboratory please contact 4 All locations except USA Email tech support lgcgroup com Phone 44 0 1992 476 486 USA only Email us support lgcgroup com Phone 1 978 338 5317 5 Protocol also available in outline form on our website THE VOLUMES OF EACH BUFFER USED IN THE PROTOCOL ARE SCALABLE IN PROPORTION TO THE AMOUNT OF STARTING MATERIAL EXTRACTED THE VOLUMES LISTED ARE FOR MAXIMUM YIELDS 5 1 Cell lysis Before you start please read the information section relevant to your sample type Section 6 1 Leaf extractions Section 6 2 Seed extractions 1 Grind dry tissue samples until homogenised a maximum of 20 30 mg material is recommended For fresh or frozen samples start immediately
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