Kleargene tissue plate DNA extraction kits user manual
Contents
1. Phone 1 978 338 5317 7 Troubleshooting Problem Blocked filter plate Low DNA yield Likely cause Excessive starting material Debris carryover from lysate Insufficient disruption of tissue prior to addition of buffer T1 Explanation suggestions Use less starting material in future extractions Ensure lysate is fully cleared via centrifugation before adding it to the filter plate wells Avoid transferring fat and other debris from the lysed centrifuged samples into the spin plate Ensure that tissue samples are ground homogenised before use Insufficient lysis Ensure that the amount of tissue used is within the recommended range up to 30 mg tissue Excess starting material will reduce the ability of cells to lyse Insufficient starting material will result in lower than expected yields Buffer E1 not pre warmed to 55 C before use DNA elution from the filter plate is much more efficient when buffer E1 is at 55 C DNA is degraded Poor sample storage prior to extraction Ensure that tissues are stored appropriately to minimise DNA degradation It is recommended that all tissue samples should be stored at 20 C or lower Precipitate has formed in buffer TO lysis buffer Incubate buffer at 37 C and mix well until clear DNA does not perform well in downstream experiments Ethanol carryover Ensure that the spin plate is completely dry after the final wash2. 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 SEE ADAPTING THE PROTOCOL TO YOUR LABORATORY UNDER NOTES 5 1 Cell lysis 1 Add 75 uL buffer T1O to the sample a maximum of 20 30 mgs material is recommended 2 Incubate the sample at 55 C until the tissue has maximally disrupted Suggested timescale 1 12 hours with certain tissue types debris may still be present afterwards 3 Add 150 uL buffer C1 to each lysed sample and mix until the solution is homogenous 4 Incubate samples at 55 C for 10 minutes 5 Centrifuge your samples at 3 000 x g for 2 minutes to clear the mixtures of most cellular debris 5 2 Absorption of DNA to glass fibre membranes 6 Place the filter plate on top of another deep well plate or reservoir Use a plate with a capacity of at least 300 uL well or an equivalent reservoir 7 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 plate The entire mixture can be transferred if it is available 8 Incubate the filter plates for 2 minutes at room temperature then centrifuge the filter plate on top of the deep well collec
3. at 37 C if this is the case and mix well until clear Ensure that the buffers are mixed well before use following the addition of the necessary reagents Component Reagent to add 1x 96 4x96 16 x 96 64 x 96 Buffer T1 O Proteinase K 7 5 mg 30 mg 125 mg 500 mg Buffer C169 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 2 Reagents to be added to buffers The volumes of each reagent to add are also declared on the labels of the bottles in the 4x and 16x kits space constraints prevent this on the bottles of the 1x kit Buffer T10 This buffer may require the addition of Proteinase K before use suggested concentration 0 5 mg mL It may be possible to reduce eliminate the Proteinase K depending on the tissue type the feasibility of this must be determined by experimentation e It is recommended that any buffer T1 O to which Proteinase K has been added should be used immediately T1 O should be aliquoted appropriately if not to be used in its entirety Buffer C1 This buffer requires the addition of 100 B mercaptoethanol before use e It is recommended that any buffer to which B mercaptoethanol is added should be used immediately C1 should be aliquoted appropriately if not to be used in its entirety Buffer A1 and W1 Buffer A1 requires the addition of etha
4. step with ethanol Salt carryover Ensure that the wash buffers A1 and W1 are at room temperature before use 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 or too little DNA can adversely affect experimental performance 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 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 nO 250 mL KBS 1012 312 c1 500 mL KBS 1012 308 me 250 mL KBS 1012 309 wie 75 mL KBS 1012 310 SK 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 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 extra
5. 12 400 KBS 1012 401 KBS 1012 442 Buffer T1 O 20 25 C 15 mL 60 mL 250 mL Buffer C1 20 25 C2 30 mL 125 mL 500 mL Buffer A1 20 25 C 15 mL 60 mL 250 mL Buffer W1 20 25 C 4 5 mL 18 mL 75 mL Buffer E1 20 25 C 15 mL 60 mL 250 mL Filter plate 20 25 C 1x96 4x 96 16 x 96 KBS 1012 443 1000 mL 2 x 1000 mL 2 x 500 mL 300 mL 1000 mL 64 x 96 Table 1 Contents of the Kleargene Tissue plate DNA extraction kit 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 e Ethanol e Isopropanol propan 2 ol B mercaptoethanol 2 mercaptoethanol e Proteinase K a stock solution should be made in an appropriate storage buffer Equipment needed e Tissue homogenisation apparatus and consumables e Manual pipettes and disposable pipette tips OR a microplate dispensing robot e Polypropylene storage plates and optionally reservoir plates e Fan oven e Waterbath incubator e Personal protective equipment lab coat gloves goggles 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 3 Reagent preparation Precipitates can form in the lysis and binding buffers after prolonged low temperature storage incubate
6. Kleargene tissue 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 Notes 6 1 General points 6 2 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 different sources The Kleargene tissue plate kit is designed for the extraction of DNA from animal tissue 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 plates of samples can be extracted in under an hour 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 2 Kit contents Component Storage KBS 10
7. ctions from plant tissues and utilises Kleargene chemistry 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 Qlgcgroup com Phone 1 978 338 5317 For any queries about this guide please contact All locations except USA email tech support Igcgroup com or call 44 0 1992 476 486 USA only email us support lgcgroup com or call 1 978 338 5317 www Igcgroup 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
8. ifuge 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 Elution of purified DNA Replace the deep well collection plate reservoir used up to this point with a clean collection plate and place the filter plate back on top of it Add 75 yL buffer E16 pre warmed to 55 C to each sample well on the filter plate e N B Yield is significantly reduced if the elution buffer is not pre warmed to 55 C 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 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 Notes 6 1 For any queries on this manual or running KASP reactions in your laboratory please contact General points The protocol is applicable in principle to any animal tissue however the protocol was developed and tested on mouse tail samples DNA is not significantly fragmented by this 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 Adapting the protocol to your laboratory If necessary the extraction process can be suspended after an
9. nol and isopropanol Buffer W1 requires the addition of ethanol only Itis not necessary to aliquot these buffers before use however it is 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 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 4 Safety information DO NOT ADD BLEACH OR ACIDIC SOLUTIONS DIRECTLY TO THE SAMPLE PREPARATION WASTE 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 the 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 5 All locations except USA Email tech support lgcgroup com Phone 44 0 1992 476 486 USA only
10. tion plate or reservoir at 3 000 x g for 2 minutes 9 Discard the supernatant from the collection plate reservoir and place the filter plate back on top of it 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 5 3 10 11 12 13 14 15 16 17 18 5 4 19 20 21 For any queries on this manual or running KASP reactions in your laboratory please contact Contaminant removal through washing Add 150 uL buffer A1 to each sample well on the filter plate 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 Discard the supernatant from the collection plate reservoir and place the filter plate back on top of it Add 75 uL buffer W1 to each sample well on the filter plate 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 Discard the supernatant from the collection plate reservoir and place the filter plate back on top of it Add 75 uL 100 ethanol to each sample well on the filter plate Incubate the filter plates for 2 minutes at room temperature then centr
11. y washing step and the plates can be kept for many hours at room temperature e If the protocol is halted for an extended period of time at any point before step 7 the lysate mixtures should be refrigerated subsequently 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 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 essentially all the remaining DNA if maximal yield is required This will inevitably reduce the overall concentration of the eluted DNA 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 T1 2 volumes C1 2 volumes A1 1 volume W1 1 volume EtOH e Some tissues occlude a certain proportion of buffer T1 O such that not all 75 HL is available for downstream processing It is acceptable to add additional TiO 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 All locations except USA Email tech support lgcgroup com Phone 44 0 1992 476 486 USA only Email us support lgcgroup com
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