CelluLyser™ Lysis and cDNA Synthesis Kit
Contents
1. ment in Appendix page 13 17 should be followed where the procedure is optimized for efficient removal of residual DNA CelluLyser Lysis and cDNA Synthesis Kit A Cell lysis The protocol is optimized for use with 1 10 000 cells Using gt 10 000 cells may result in incomplete lysis and or inhibition of cDNA synthesis The cell lysis can be performed in either 0 2 ml PCR tubes 384 well cell culture plates or in 96 well cell culture plates Up to 2 000 cells may be lysed in 5 5 ul lysis solution For larger number of cells 2 000 10 000 the lysis solution volume is scaled up to 13 75 ul Additionally if using a 96 well cell culture plate a lysis solution volume of 13 75 ul must be used to cover the well surface properly 1 Prepare a lysis solution Add CelluLyser buffer and Protector RNase inhibitor to a tube or well accord ing to the table below See above for guidelines about the lysis solution volume depending on cell amount and cell vessel formats A master mix may be prepared for multiple reactions Use the CelluLyser buffer equilibrated to room temperature Lysis solution for 1rxn Component 1 2 000 cells 2 000 10 000 cells or 96 well cell culture plate CelluLyser buffer Sul 12 5 ul Protector RNase inhibitor 0 5 ul 1 25 ul Total volume 5 5 pl 13 75 ul 2 Mix lysis solution and cells Single cells Patch clamp collection FACS sorting etc Collect the cell with a minimal amount of residual volume and2. The entire cDNA synthesis reaction can be transferred from one sample vessel to another If using a well plate seal it hermetically using for example a sealing foil This is critical to avoid evaporation during cDNA synthesis If us ing a tube make sure the lid is properly closed Incubate the plate or the tube according to the temperature proto col below If using a heating cabinet make sure that it is pre heated before each incubation step and cool the reaction to 4 C by putting it on ice 85 C 5 min 50 C 30 min 25 C 10 min BL Se Hold If the heating cabinet cannot be heated to 85 C this step can be replaced with an incubation at 70 C for 10 minutes At this point the synthesized cDNA may be stored at 2 to 8 C for several hours or at 15 to 25 C for longer periods The cDNA can be added toa PCR reaction without purification Note Add maximum 10 of un diluted cDNA in the PCR reaction to avoid inhibiton e g 2 ul to a 20 ul reaction CelluLyser Lysis and cDNA Synthesis Kit Troubleshooting For problems occuring during PCR see the instructions for the PCR kit you are using Problems occuring during lysis and cDNA synthesis can be Excessive cell number gt 10 000 cells Make sure an amount of 1 10 000 cells is used If using gt 10 000 cells it may result in incomplecte lysis and or inhibiton of cDNA synthesis Insufficient washing of cells To avoid incomplete lysis make sure cells are caref
3. add it directly to the lysis solution During the collection the cell should be kept in a medium or buffer that is not inhibitory to enzymatic reactions e g 1X PBS buffer Multiple cells may be added to the tube or well as long as the residual volume is low enough to avoid any significant dilution of the lysis solution which may cause incomplete lysis of the cells Cellsinsolution a Pellet the cells and wash the pellet with 4 C 1X PBS buffer b Carefully remove the PBS buffer without disturbing the cells It is critical to remove as much PBS buffer as possible as remaining buffer will dilute the lysis solution and may cause incomplete lysis c Dissolve the cell pellet in lysis solution Adherent cells grown in 96 or 384 well cell culture plates a Aspirate the culture media from each well Wash the cells with 4 C 1X PBS buffer b Carefully remove the PBS buffer without disturbing the cells It is critical to remove as much PBS buffer as possible as remaining buffer will dilute the lysis solution and may cause incomplete lysis c Add lysis solution to each well 3 Lysis of cells Incubate the cells in lysis solution at room temperature for 10 minutes Continue with B cDNA synthesis on next page or store the lysate until the sam ple can be processed The lysate may be stored at 4 C for up to two weeks or at 70 C for long term storage CelluLyser Lysis and cDNA Synthesis Kit B cDNA synthesis cDNA synthesis is perf
4. an indication of either presence of genomic DNA or contami nating DNA in the sample Either perform the optional DNase treatment or consider measures of DNA contamination of reagents pipettes benchtops etc CelluLyser Lysis and cDNA Synthesis Kit Appendix Optional DNase treatment A separate DNase treatment to degrade contaminating genomic DNA is of ten not required with the CelluLyser Lysis and cDNA Synthesis Kit The Cel luLyser buffer lyses the cells gently releasing RNA completely from the cells without making the genomic DNA fully accessible to the DNA polymerase The level of genomic DNA contamination and the effect on gene expression analysis should be assessed for each gene using NoRT controls A NoRT control contains all the cDNA synthesis components including lysate except the Re verse Transcription enzyme substitute with water If a NoRT control gives an amplification curve when performing downstream PCR the signal is caused by amplification of genomic DNA A rule of thumb is that the Cq value of the NoRT control should be at least 5 cycles higher than the Cq value of the sample that is being quantified If the Cq value of the NoRT control is too low a DNase treatment of the lysate is needed Residual DNA can be removed with the optional DNase treatment pro cedure using the double strand specific DNase dsDNase ArcticZymes Please note that DNase is not included in the kit Procedure overview Lysis 1 10
5. reactions are depending on cell amount and vessel format License information The purchase of this product conveys to the buyer the non transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer whether the buyer is an academic or for profit entity The buyer cannot sell or otherwise transfer a this product b its components or c materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for commercial purposes For information on purchasing a license to this product for purposes other than research contact TATAA Biocenter AB Odinsgatan 28 S 41103 G teborg Sweden Phone 46 31 761 57 00 Fax 46 31 152890 Email info tataa com PCR is covered by several patents owned by Hoffman La Roche Inc and Hoffman LaRoche Ltd Purchase of the product does not include or provide a license with respect to any PCR related patent owned by Hoffman La Roche or others TATAA Biocenter does not encourage or support the unauthorised or unlicensed use of the PCR process CelluLyser Lysis and cDNA Synthesis Kit Other products from TATAA dsDNase DNase enzyme from ArcticZymes which is specific to double stranded DNA Double strand specific nuclease is efficiently inactivated at 65 C and can be included in your RT reaction without degrading the single stranded cDNA HL d
6. 000 cells are lysed in CelluLyser buffer at room temperature for 10 min utes and heated to 85 C for 5 minutes to make the genomic DNA accessible for DNase treatment Depending on the amount of cells or the cell vessel format the lysis volume is 5 ul or 12 5 ul Thereafter RNase inhibitors are added cDNA synthesis reaction including DNase is prepared A master mix for the cDNA synthesis including the dsDNAse is prepared ap propriate for the cell lysate volume cDNA synthesis and DNase treatment The cDNA synthesis and simultaneous DNase treatment are performed either in a heating cabinet or in a thermocycler with a heated lid Ready cDNA The synthesized cDNA may be stored or added to a PCR reaction without pu rification 13 14 A Cell lysis The protocol is optimized for use with 1 10 000 cells Using gt 10 000 cells may result in incomplete lysis and or inhibition of cDNA synthesis The cell lysis can be performed in either 0 2 ml PCR tubes 384 well cell culture plates or in 96 well cell culture plates Up to 2 000 cells may be lysed in 5 ul CelluLyser buffer For larger number of cells 2 000 10 000 the CelluLyser buffer volume is scaled up to 12 5 ul Ad ditionally if using a 96 well cell culture plate a CelluLyser buffer volume of 12 5 ul must be used to cover the well surface properly 1 Mix CelluLyser buffer and cells Mix pure CelluLyser buffer and cells in a tube or well according to description and add th
7. User Manual CelluLyser Lysis and cDNA Synthesis Kit Version 1 4 Oct 2012 From cells to cDNA in one tube Q tataabiocenter CelluLyser Lysis and cDNA Synthesis Kit Table of contents troduction eea e a e a Contents E E toca E E E Storage e D Additionally required materials and devices E Safety information en PP A indeed acto elas Syed a norssmtircn a de Procedure overview ERE A E cai E E E A B cDNA synthesis I Troubleshooting reeeo eea Appendix Optional DNase treatment ccc otal Procedure OVERVIEW sorcen al ACENSIS Eere a a lA B cDNA synthesis and DNase treatment 16 Contact and ordering information nd 8 License information coe 18 Other products from TATAA oe an es 19 Introduction The CelluLyser cDNA Synthesis Kit has been developed for fast and simple cell lysis and cDNA synthesis of small samples With the possibility to generate cDNA directly from cell lysate without isolating RNA considerable amount of time is saved compared to standard RNA purification The kit comprises the CelluLyser buffer for a rapid and sensitive lysis of cells and cDNA synthesis reagents for reverse transcription of RNA into cDNA for samples ranging from 10 000 cells down to as little as one single cell To avoid losses of material the entire procedure can be performed in a single tube without any sample trans fer Also 100 of the total RNA content may be used from lysis to cDNA syn thesis T
8. e buffer volumes stated in the table below See A Cell lysis above for guidelines about the buffer volume depending on cell amount and cell vessel formats Use the CelluLyser buffer equilibrated to room temperature Do not add Protector RNase inhibitors at this stage Lysis buffer for 1rxn Component 1 2 000 cells 2 000 10 000 cells or 96 well cell culture plate CelluLyser buffer 5 ul 12 5 ul Single cells Patch clamp collection FACS sorting etc Collect the cell with a minimal amount of residual volume and add it directly to the CelluLyser buffer During the collection the cell should be kept in a medium or buffer that is not inhibitory to enzymatic reactions e g 1X PBS buffer Multiple cells may be added to the tube or well as long as the residual volume is low enough to avoid any significant dilution of the CelluLyser buffer which may cause incomplete lysis CelluLyser Lysis and cDNA Synthesis Kit Cells in solution a Pellet the cells and wash the pellet with 4 C 1X PBS buffer b Carefully remove the PBS buffer without disturbing the cells It is critical to remove as much PBS buffer as possible as remaining buffer will dilute the CelluLyser buffer and may cause incomplete lysis c Dissolve the cell pellet in CelluLyser buffer Adherent cells grown in 96 or 384 well cell culture plates a Aspirate the culture media from each well Wash the cells with 4 C 1X PBS buffer b Carefully remove the PBS buffer without dist
9. ered from TATAA Biocenter with the kit 1 Thaw all frozen reagents before use Briefly centrifuge them before starting the procedure 2 Prepare a cDNA synthesis master mix including DNase according to one of the tables below depending on the cell lysate volume Prepare the master mix appropriate for the number of samples you are processing Mix the reagents gently Do not vortex Mastermix for 5 5ul cell lysate Component 1rxn Final conc dsDNAse 2 U ul Tul 0 1 U ul Anchored oligo dT Primer Tul 2 5 uM Random Hexamer Primer 2 ul 60 uM Water PCR grade 4ul Transcriptor RT Reaction Buffer 5X 4ul 1X Deoxynucleotide Mix 2ul 1 mM each Transcriptor Reverse Transcriptase 0 5 ul 0 5 U ul Final mastermix volume 14 5 ul Mastermix for 13 75 ul cell lysate Component Final conc dsDNAse 2 U ul 2 5 ul 0 1 U ul Anchored oligo dT Primer 2 5 ul 2 5 uM Random Hexamer Primer Sul 60 uM Water PCR grade 10 ul Transcriptor RT Reaction Buffer 5X 10 ul 1X Deoxynucleotide Mix Sul 1 mM each Transcriptor Reverse Transcriptase 1 25 ul 0 5 U ul Final mastermix volume 36 25 ul CelluLyser Lysis and cDNA Synthesis Kit Add the cDNA synthesis master mix to each tube or well either 14 5 ul to 5 5 ul cell lysate final volume 20 ul or 36 25 ul to 13 75 ul cell lysate final volume 50 ul Mix gently by pipetting Do not vortex Note A maximum of 5 5 ul lysis solution can be added to a 20 ul cDNA synthesis reaction cDNA synthesis may be p
10. erformed either in a heating cabinet or in a thermocycler with a heated lid The entire cDNA synthesis reaction can be transferred from one sample vessel to another If using a well plate seal it hermetically using for example a sealing foil This is critical to avoid evaporation during cDNA synthesis If using a tube make sure the lid is properly closed Incubate the plate or the tube according to the temperature proto col below If using a heating cabinet make sure that it is pre heated before each incubation step and cool the reaction to 4 C by putting it on ice 85 C 5min 50 C 30 min 37 C 20 min Be SSS 4 c Hold 25 C 10 min BL If the heating cabinet cannot be heated to 85 C this step can be replaced with an incubation at 70 C for 10 minutes At this point the synthesized cDNA may be stored at 2 to 8 C for several hours or at 15 to 25 C for longer periods The cDNA can be added to a PCR reaction without purification Note Add maximum 10 undiluted DNA to the PCR reaction i e 2 ul to a 20 ul reaction to avoid inhibition 17 18 Contact and ordering information To reorder or for more information about the product and other products available from TATAA Biocenter please contact us on order tataa com or visit our website www tataa com CelluLyser Lysis and cDNA Synthesis Kit CelluLyser buffer 250 or 100 rxns and Order H101 cDNA synthesis reagents 100 or 40 rxns the amount of
11. he CelluLyser procedure is rapid and straightforward Cells are lysed in only 10 minutes in the optimized CelluLyser buffer and thereafter sensitive cDNA synthesis is performed directly on the cell lysate I Lysis 10 mins I cDNA synthesis Mix lysis solution Add mastermix for cDNA and cells cDNA synthesis ready for PCR CelluLyser Lysis and cDNA Synthesis Kit Contents The reagents provided are sufficient for 250 lysis reactions and 100 cDNA syn thesis reactions of 1 2 000 cells 20 ul cDNA synthesis reactions or 100 lysis reactions and 40 cDNA synthesis reactions of 2 000 10 000 cells 50 ul cDNA synthesis reactions See A Cell lysis on page 7 and B cDNA synthesis on page 9 for more information Component Volume Concentration CelluLyser buffer 1 25 ml 1X cDNA synthesis reagents Roche Transcriptor First Strand cDNA Synthesis Kit Transcriptor Reverse Transcriptase 50 ul 20 U ul Transcriptor RT Reaction Buffer 1ml 5X Protector RNase Inhibitor 100 ul 40 U ul Deoxynucleotide Mix 200 ul 10 mM each Anchored oligo dT Primer 200 ul 50 uM Random Hexamer Primer 200 ul 600 uM Water PCR grade 2x1 ml Storage The CelluLyser buffer can be stored at 4 C for 12 months Store the cDNA syn thesis reagents at 15 to 25 C through the expiration date printed on the label Avoid repeated freeze thaw cycles Additionally required materials and devices General laboratory equipment including pipettes and microcent
12. ormed using the Transcriptor First Strand cDNA synthesis kit Roche 1 Thaw all frozen reagents before use Briefly centrifuge them before starting the procedure Prepare a cDNA synthesis maste rmix according to one of the tables below depending on the cell lysate volume Prepare the master mix appropriate for the number of samples you are processing Mix the reagents gently Do not vortex Mastermix for 5 5 ul cell lysate Component Irxn Final conc Anchored oligo dT Primer Tul 2 5 uM Random Hexamer Primer 2ul 60 uM Water PCR grade Sul Transcriptor RT Reaction Buffer 5X 4ul 1X Deoxynucleotide Mix 2 ul 1 mM each Transcriptor Reverse Transcriptase 0 5 ul 0 5 U ul Final master mix volume 14 5 ul Mastermix for 13 75 pl cell lysate Component Irxn Final conc Anchored oligo dT Primer 2 5 ul 2 5 uM Random Hexamer Primer 5 ul 60 uM Water PCR grade 12 5 ul Transcriptor RT Reaction Buffer 5X 10 ul 1X Deoxynucleotide Mix 5 ul 1 mM each Transcriptor Reverse Transcriptase 1 25 ul 0 5 U ul Final master mix volume 36 25 ul 10 Add the cDNA synthesis master mix to each tube or well either 14 5 ul to 5 5 ul cell lysate final volume 20 ul or 36 25 ul to 13 75 ul cell lysate final volume 50 ul Mix gently by pipetting Do not vortex Note A maximum of 5 5 ul lysis solution can be added to a 20 ul cDNA synthesis reaction cDNA synthesis may be performed either in a heating cabinet or in a thermocycler with a heated lid
13. real time PCR expression panels TATAA Biocenter has great experience and expertise in high resolution gene expression profiling pathogen de tection and small sample single cell analysis TATAA Biocenter AB Odinsgatan 28 411 03 G teborg Tel 46 31 761 57 00 Fax 46 31 15 28 90 E mail info tataa com Website www tataa com
14. rifuge Nuclease free pipette tips and microcentrifuge tubes Heating cabinet and ice or thermocycler 4 C 1x phosphate buffered saline PBS for cells requiring washing For optional DNase treatment procedure dsDNase or HL dsDNase from ArcticZymes Safety information When working with chemicals always wear a protective lab coat disposable gloves and protective eyewear See the appropriate material safety sheets MSDSs for more information Procedure Procedure overview Lysis 1 10 000 cells are lysed in lysis buffer containing RNase inhibitors at room tem perature for 10 minutes Depending on the amount of cells or the cell vessel format the lysis volume is 5 5 ul or 13 75 ul cDNA synthesis reaction is prepared A mastermix for the cDNA synthesis is prepared appropriate for the cell lysate volume cDNA synthesis The cDNA synthesis is performed either in a heating cabinet or in a thermocy cler with a heated lid Ready cDNA The synthesized cDNA may be stored or added to a PCR reaction without pu rification Optional DNase treatment A separate DNase treatment to degrade contaminating genomic DNA is often not required with the CelluLyser Lysis and cDNA Synthesis Kit The CelluLys er lysis buffer lyses the cells gently releasing RNA completely from the cells without making the genomic DNA fully accessible to the DNA polymerase If a DNase treatment of the lysate is desired the protocol Optional DNase treat
15. sDNase New generation DNase enzyme from ArcticZymes which is specific to double stranded DNA Heat Labile double strand specific nuclease is efficiently inacti vated at 55 C and can be included in your RT reaction without degrading the single stranded cDNA Reference Gene Panel Human or Mouse The panel contains primer sets for 12 commonly used human or mouse refer ence genes A perfect product for fast detection and confirmation the most op timal reference genes for your samples A one year licence for GenEx Standard software with GeNorm and Normfinder is included in the kit GenEx software A software developed for gene expression analysis GenEx provides the appro priate tools to analyze real time PCR gene expression data and to extract valu able information from the measurements VisiBlue qPCR mix colorant The VisiBlue qPCR mix colorant enables you to quickly color your favourite qPCR mastermix to easily visualize where the reagent has been added to your plates and tubes VisiBlue is easy to use by a simple addition to your master mix 19 Express your genius TATAA Biocenter with offices in Gothenburg San Francisco and Prague is the leading provider of real time PCR services and the prime organizer of real time PCR work shops globally TATAA Biocenter con ducts commissioned research and training within the field of molecu Q tataabiocenter lar diagnostics and gene expression analysis along with developing
16. ully washed with PBS and also make sure a minimal amount of residual volume is added to the lysis solution Insufficient volume of lysis buffer Up to 2 000 cells may be lysed in 5 5 ul lysis solution For larger num ber of cells 2 000 10 000 the lysis solution volume must be scaled up to 13 75 ul Additionally if using a 96 well cell culture plate a lysis solution volume of 13 75 ul must be used to cover the well surface properly Insufficient volumes of cDNA synthesis reagents MakesurethevolumesofthecomponentsaddedtothecDNA synthesis master mix are correct 14 5 ul master mix should be prepared for 5 5 ul cell lysate final volume 20 ul or 36 25 ul for 13 75 ul cell lysate final volume 50 ul A maximum of 5 5 ul lysis solution can be added to a 20 ul cDNA synthesis reaction Lysis and cDNA synthesis are not performed in referred duration times or temperatures Make sure the CelluLyser buffer has been equilibrated to room tem perature and that the incubation of the cells in lysis solution is perfor med at room temperature for 10 minutes For the cDNA synthesis incubate the plate or the tube according to the stated temperature protocol 11 12 The lysate or the cDNA are not stored properly The lysate may be stored at 4 C for up to two weeks or at 70 C for long long term storage The synthesized cDNA may be stored at 2 to 8 C for several hours or at 15 to 25 C for longer periods Positive NoRT control This is
17. urbing the cells It is critical to remove as much PBS buffer as possible as remaining buffer will dilute the CelluLyser buffer and may cause incomplete lysis c Add CelluLyser buffer to each well 2 Lysis of cells Incubate cells in CelluLyser buffer at room temperature for 10 minutes fol lowed by heating to 85 C for 5 minutes to make the genomic DNA accessible for DNase treatment Cool the sample to 4 C 3 Addition of RNase inhibitors Add RNase inhibitors according to the table below Make sure the sample has been cooled to 4 C before addition RNase inhibitors for 1rxn Component 5 ul CelluLyser buffer 12 5 ul CelluLyser buffer Protector RNase inhibitor 0 5 ul 1 25 ul Total lysate volume 5 5 ul 13 75 ul Continue with B cDNA synthesis and DNase treatment on next page or store the lysate until the sample can be processed The lysate may be stored at 4 C for up to two weeks or at 70 C for long term storage 15 16 B cDNA synthesis and DNase treatment cDNA synthesis is performed using the Transcriptor First Strand cDNA synthesis kit Roche The simultaneous DNase treatment is performed using a double strand specific DNase dsDNAse ArcticZymes which is specially designed for DNase treatment during the RT step The nuclease shows 30x higher activity than bovine DNAse and the activity towards dsDNA is 5000x higher than to wards ssDNA Please note that DNase is not included in the kit but can be or d
Download Pdf Manuals
Related Search