PicoPLEX™ DNA-seq Kit Quick Protocol
Contents
1. Kit Instruction Manual B Additional materials and equipment needed Thermal cycler centrifuge PCR tubes or plates PCR plate seals low binding barrier tips fluorescent dyes 10X Phosphate Buffered Saline 10X PBS buffer free of Meg Ca and BSA 80 v v Ethanol C Notes before starting 1 Starting material requirements Mammalian cells 1 10 cells or isolated DNA 15 60 pg Single mammalian cells from sources such as embryos tumor cells and cultured clonally expanded cells E Single cells obtained by micro manipulation and flow sorting as well as flow sorted cells that are stained with surface antibodies are also suitable Avoid cell fixation for optimal results 2 Washing the cells Wash the cells with sterile nuclease free 1X PBS freshly prepared from a 10X PBS stock Carryover PBS volume must not exceed 2 5 uL 3 Selecting PCR Plates Tubes Select plates tubes that are compatible with the thermal cyclers and or real time PCR instruments used Ensure that there is no evaporation during the process by using proper seal caps during cycling as evaporation may reduce robustness and reproducibility 4 Positive and Negative Controls Including a positive control DNA 15 pg and a No Template Control NTC as a negative control in parallel is recommended to ensure that the reaction proceeded as expected 5 Preparation of Master Mixes Keep all enzymes and buffers on ice Always prepare a master mix immediately prior to use2. Orange 25 0 UL Amplification Enzyme Blue 0 5 UL Fluorescent Dyes 2 5 UL Nuclease Free Water Clear Varies Total 30 UL Reaction Fluorescence dyes detection and optical calibration are added when monitoring the amplification in real time during cycling Please refer to the Real Time PCR Instrument s user manual for calibration dye recommendations and add an appropriate amount of the detection and calibration dye If a regular thermal cycler is used there is no need to add the dyes substitute with appropriate amount of nuclease free water instead to adjust the final volumes Example EvaGreen Fluorescein dye mix Prepare by mixing 9 1 v v ratio of 20X EvaGreen dye Cat 31000 T 20X in water Biotium Inc and 1 500 diluted Fluorescein Fluorescein Calibration Dye Catalog 170 8780 Bio Rad Laboratories add 2 5 uL of this mix per reaction Remove the seal on the PCR plate and add 30 uL of the amplification master mix to each well Add 5 uL of the appropriate index combination from the index plate to each well and mix gently several times with a pipettor avoiding introducing excessive air bubbles Follow the index plate handling instructions C 8 to avoid index cross contamination Note Final volume at this stage is 50 uL Seal the plate tightly and centrifuge briefly to collect the contents to the bottom of each well Return plate to the real time PCR cycler thermal cycler with a heated lid set to 105 C and perform Amplificatio
3. PicoPLEX DNA seq Kit Quick Protocol PicoPLEX DNA seq Kit CAT NO R300381 48 reactions is intended for amplifying genomic DNA from single cells to reliably detect chromosomal aneuploidies and copy number variations CNV on Illumina s Next Generation Sequencing NGS platforms Applications include pre implantation genetic screening PGS using blastomeres and trophectoderm cells Other applications include NGS based CNV detection in Circulating Tumor Cells CTCs where single cell amplifications are required For more information visit www rubicongenomics com products PicoPLEX DNA seq For detailed information please refer to the PicoPLEX DNA seq Kit Instruction Manual at www rubicongenomics com resources manuals Storage and Handling Store the kit at 20 C upon arrival Transfer enzymes to ice prior to use and centrifuge briefly Thaw the buffers vortex briefly and centrifuge prior to use Store on ice until used Kit Contents PicoPLEX DNA seq Dual Indexes Dual Index Plate Index i7 S Name Cap Color Pre Amp Buffer Red Blue i5 Index Sequence i7 Index Sequence 0502 0593 05 050 a x g c e D596 0507 s 000000000000 A Kit Contents M PicoPLEX DNA seq Kit is provided with one Dual Index Plate and contains sufficient reagents see Kit Contents table above to accommodate 48 reactions at once or up to 4 uses of the plate as described in the PicoPLEX DNA seq
4. and keep on ice while pipetting Assembling the reactions on ice is recommended 6 Dual Index Plate PicoPLEX DNA seq Kit is provided with a Dual Index Plate containing 48 Illumina compatible dual indexes Each well contains a unique combination of Illumina s 8nt TruSeq HT i5 and i7 index sequences see table above middle The index combination at each well position is indicated by the column i7 and row i5 labels on the plate map see figure above right E Wells corresponding to columns 7 to 12 are empty M Each well contains sufficient volume of dual index combination for single use This plate is sealed with pierceable sealing foil Refer to the plate handling instructions below for more information 7 Low level multiplexing This kit is designed for high throughput applications however it can also be used for low level multiplexing of smaller numbers of samples The Index Plate can be frozen and thawed no more than four times Select appropriate dual index combinations that meet Illumina recommended compatibility requirements The well colors of the plate map see figure above right illustrate one way to pool dual index combinations for an 8 plex experiment wells sharing the same color should be pooled together For other ways to pool a low plex 2 to 16 plex experiment please refer to Illumina s TruSeq Sample Preparation Pooling Guide Illumina Part 15042173 Rev B 2014 8 Index Plate Handling Instructions Follow the instr
5. fly to ensure entire volume of the reaction is collected at bottom of each well 4 Return the plate to the thermal cycler with a heated lid set to 105 C and perform Pre Amplification Reaction using the cycling conditions from in the table below Pre Amplification Reaction Step Temperature Time Number of cycles Step 1 95 C 2 min 1 cycle 95 C 15 sec l5 C 50 sec 25 C 40 sec Step 2 12 l E e 30 sec e 65 C 40 sec 75C 40 sec Step 3 4 C Hold 1 cycle 5 Remove the plate and centrifuge briefly continue to the Library Amplification Step in the same plate For technical support contact support rubicongenomics com or call 734 677 4845 PicoPLEX M_DNA seq kit is for research use only It may not be used for any other purposes including but not limited to use in diagnostics forensics therapeutics or in humans PicoPLEX DNA seq may not be transferred to third parties resold modified for resale or used to manufacture commercial products without prior written approval of Rubicon Genomics Inc PicoPLEX DNA segq is protected by US Patent 8 206 913 other US and foreign patents pending Trademarks PicoPLEX Bio Rad Bio Rad laboratories Inc illumina Illumina Inc Eva Green dye Biotium Inc J4 Library Amplification Step Prepare Amplification Master Mix as described in the table below Store on ice until used Amplification Master Mix Component Cap Color Volume Reaction Amplification Buffer
6. n Reaction using the cycling conditions from the table below Amplification Reaction Step Temperature Time Number of cycles Step 1 95 C 4 min 1 cycle 95 C 20 sec Step 2 63 C 25 sec A cycles 72 C AO sec 95 C 20 sec P Step 3 72 C 55 sec 7 cycles Step 4 4 C Hold Acquire fluorescence data at this step if monitoring amplification in real time At the end of library amplification remove the plate from the cycler and centrifuge briefly At this stage samples can be processed for library purification immediately or stored frozen at 20 C and processed later Refer to the detailed user manual available online for recommendations on library purification subsequent quantification and next generation sequencing NGS EA RUBICON GENOMICS
7. recommended 2 Depending on the number of reactions prepare Enzyme Extraction Master Mix as described in the table below Mix gently several times Enzyme Extraction Master Mix Component Cap color Volume Reaction Extraction Enzyme Dilution Buffer wiciet ene Cell Extraction Enzyme Yellow 0 2 UL 3 To each 5 uL equilibrated sample from step 1 above add 5 uL of the Enzyme Extraction Master Mix taking care not to disturb cell E Note Final volume at this stage will be 10 uL Seal the PCR plate using proper sealing film and centrifuge briefly to ensure entire volume of the reaction is collected at bottom of each well 5 Place the plate in a thermal cycler with a heated lid set to 105 C and perform the Lysis Reaction using the protocol in the table below Lysis Reaction Temperature Time 75 GE 10 min 95 C A min 22 C Hold 6 Remove the plate and centrifuge briefly continue to the Pre Amplification Step in the same plate ll Pre Amplification Step _ Prepare a pre amplification master mix as described in the table below Mix gently several times Store on ice until used Pre Amplification Master Mix Component Cap Color Volume Reaction Pre Amp Buffer Red 4 8 UL Pre Amp Enzyme White 0 2 UL 2 Remove the seal on the plate add 5 uL of the pre amplification master mix to each well E Note Final volume at this stage is 15 uL 3 Seal the PCR plate using proper sealing film and centrifuge brie
8. uctions given below to avoid potential index cross contamination Thaw the index plate for 10 min on the bench top prior to use Once thawed briefly centrifuge the plate to collect the contents to the bottom of each well Thoroughly wipe the foil seal with 70 ethanol and allow it to dry completely Pierce the seal above the specific index combination with a 200 uL filtered pipet tip discard the tip a Use a new pipet tip to collect 5 uL of a specific index combination i5 amp i7 and add it to the reaction at the amplification step If several reactions are being assembled a multichannel pipette may be used If indexes from the entire plate are not used at the same time follow the instructions below to avoid contamination Cover any pierced index wells with scientific tape such as VWR General Scientific Tape 0 5 CAT NO 89097 920 to mark the index as used Once the dual index plate is used wipe the seal with 70 ethanol and let it dry completely Replace the plastic lid and return the plate to its sleeve and store at 20 C AN Sie QAM I I 1 002 RUBICON GENOMICS D PicoPLEX DNA seq Quick Protocol I Cell Lysis Step 1 Prepare a PCR plate where each experimental well contains individual single cells or genomic DNA dilutions in 5 uL of buffer refer to User Manual for detailed instructions for preparing the single cell or diluted DNA samples Wells containing 5 uL of NTC negative control buffer sample s are also
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