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Thermo Scientific KingFisher Pure RNA Blood Kit, Инструкция по эксплуатации

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   Thermo Scientific KingFisher Pure RNA Blood Kit

Thermo Fisher Scientific

Chapter 5

   

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   Protocols and Pipetting Instructions 

When the KingFisher Flex pauses at the second dispense step after the 
DNase step at approximately 10 minutes after the first dispense step, 
remove the DNase plate from the instrument, and add the Rebinding Buffer 
(supplemented with ethanol) to the 

DNase plate

 to rebind the RNA.

Plate name

Add

Added reagent volume 

per well

DNase

Rebinding Buffer

490 µl

9.  Place the DNase plate back into the instrument and press 

Start

. After 

the pause, the protocol will continue to completion.

10.  When the protocol is completed, remove the plates according to the 

instructions on the KingFisher Flex display and switch off the instrument. 
The purified RNA is ready for use in downstream applications. When 
working with RNA, keep the purified samples on ice. Store the purified 
RNA at -20°C or -80°C.

NOTE:

 The final RNA concentration in the nuclease-free water may 

increase if the purified RNA is eluted into a smaller than recommended 
volume of water, but this can slightly reduce the overall RNA yield.

Instructions for KingFisher Duo with 12-pin 

Magnet Head

These instructions are intended for RNA purification from 200 µl of blood, 
using the KingFisher Pure RNA Blood Kit (Cat. No. 98020196) and the 
KingFisher Duo with 12-pin magnet head.

When using the KingFisher Pure RNA Blood Kit for the first time, prepare 
the DNase I storage solution, Rebinding Buffer, Wash Buffer 1, and Wash 
Buffer 2. For each run, prepare the DNase I working solution and Lysis Buffer 
with 2 M DTT. For more instructions, refer to Chapter 4: 

“Storage Conditions 

and Preparation of the Reagents”

.

Check all the solutions in the kit for salt precipitation before each use. 
Redissolve precipitates by warming the solution at 37°C and equilibrate to 
room temperature (15−25°C).

1.   Take one empty Microtiter deep well 96 plate and one Thermo Scientific™ 

KingFisher™ Duo elution strip. 

Содержание KingFisher Pure RNA Blood Kit

Страница 1: ...Instruc t io n Manual Thermo Scientific KingFisher Pure RNA Blood Kit Revision 1 0 N14035 March 2013 ...

Страница 2: ...roducts are for Research Use Only Not for use in diagnostic procedures The Product will operate substantially in conformance with Thermo Fisher Scientific s published specifications THERMO FISHER SCIENTIFIC DISCLAIMS ALL OTHER WARRANTIES WHETHER EXPRESSED OR IMPLIED ORAL OR WRITTEN WITH RESPECT TO THE PRODUCTS INCLUDING WITHOUT LIMITATION ALL IMPLIED WARRANTIES OF PRODUCT QUALITY CONDITION DESCRIP...

Страница 3: ... Storage 16 Chapter 5 Protocols and Pipetting Instructions 17 Handling of KingFisher Magnetic Beads 17 Avoiding Ribonuclease RNase Contamination 17 Instructions for KingFisher Flex with 96 Deep Well Plates 18 Instructions for KingFisher Duo with 12 pin Magnet Head 20 Instructions for Total RNA Purification from PAXgene Stabilized Blood Samples 23 Instructions for Total RNA Purification from Tempus...

Страница 4: ......

Страница 5: ...ution Buffer 2 x 1 0 ml Manganese Chloride Solution 1 ml 2 x DNase I Buffer 3 x 1 5 ml Nuclease free water 30 ml Addition of ethanol or isopropanol required The KingFisher Pure RNA Blood Kit Cat No 98020196 is intended for the purification of blood samples using the Thermo Scientific KingFisher Flex with a 96 deep well head or the Thermo Scientific KingFisher Duo with a 12 pin head and a sample vo...

Страница 6: ...netic Beads at 4 C Other kit components can be stored at room temperature 15 25 C The reagents are stable for up to two years from the manufacturing date Additional Reagents Required 96 100 ethanol EtOH molecular biology grade 100 isopropanol molecular biology grade 2 M DTT dithiothreitol solution store at 20 C PBS Phosphate buffered saline for Tempus protocol Table 1 2 Thermo Scientific KingFishe...

Страница 7: ...uct Package size 97003500 KingFisher Duo 12 tip comb for Microtiter deep well 96 plate 50 pcs 97003520 KingFisher Duo elution strip 40 pcs 95040450 Microtiter deep well 96 plate 50 pcs 95040460 Microtiter deep well 96 plate sterile 50 pcs 97003530 KingFisher Duo Combi pack for Microtiter deep well 96 plate tip combs plates and elution strips for 96 samples 1 box ...

Страница 8: ......

Страница 9: ...e RNA purified using the KingFisher Pure RNA Blood Kit is of high quality and free of proteins nucleases and other contaminants or inhibitors It is therefore suitable for direct use in many different downstream applications such as RT qPCR reverse transcription quantitative PCR RT PCR and several other enzymatic reactions Intended Use The KingFisher Pure RNA Blood Kit is developed for the purifica...

Страница 10: ...aminants while the RNA bound to the KingFisher Magnetic Beads is transferred through the wash steps Two different Wash Buffers are used followed by an air drying step High quality RNA is eluted into nuclease free water and is ready for subsequent downstream processes Kit Specifications The KingFisher Pure RNA Blood Kit is designed for rapid automated preparation of highly pure total RNA from up to...

Страница 11: ...It Software 3 2 can be used for running ready made and optimized protocols for the Thermo Scientific KingFisher Pure Kits It is also possible to transfer the developed protocol onto the onboard software and run it directly from the instrument The KingFisher instruments provide a rapid and automated solution for complicated and time consuming purification processes resulting in high purity RNA with...

Страница 12: ...ex 24 deep well plate 12 pin magnet head for Microtiter deep well 96 plate 6 pin magnet head for KingFisher Flex 24 deep well plate Plates KingFisher Flex 96 KF plate 20 200 µl 96 well PCR plate skirted 20 100 µl Microtiter deep well 96 plate 50 1000 µl KingFisher Flex 24 deep well plate 200 5000 µl Microtiter deep well 96 plate 50 1000 µl KingFisher Duo elution strip 30 130 µl KingFisher Flex 24 ...

Страница 13: ...guarantee reliable analyses Table 3 1 Safety precautions Reagent Hazardous contents Safety instructions Lysis Buffer Guanidium thiocyanate Harmful by inhalation Liberates very toxic gas in contact with acids Harmful to aquatic organisms and may cause long term adverse effects in the aquatic environment Keep the container in a well ventilated place Do not breathe gas fumes vapor spray Wear suitable...

Страница 14: ...ing Buffer conc Sodium perchlorate Harmful if swallowed Explosive when mixed with combustible material Keep away from combustible material Do not breathe gas fumes vapor spray Take off immediately all contaminated clothing Wear suitable protective clothing This material and its container must be disposed of as hazardous waste Wash Buffer 1 conc Guanidium chloride Harmful if swallowed Irritating to...

Страница 15: ... at 4 C Other kit components can be stored at room temperature 15 25 C The reagents are stable for up to two years from the manufacturing date Preparation of the Lysis Buffer Before each RNA purification experiment a fresh aliquot of Lysis Buffer should be supplemented with 2 M DTT solution not provided It is required to use 200 µl of Lysis Buffer for the purification of one sample Add 20 µl of 2 ...

Страница 16: ... gentle rotation of the vial helps to dissolve the DNase I but avoid forceful mixing Store the DNase I storage solution at 20 C Repeated freezing and thawing should be avoided Preparation of the DNase I Working Solution Calculate the amount of DNase I working solution needed For the purification of one sample mix 35 µl of 2 x DNase I Buffer 1 5 µl Manganese Chloride Solution 10 µl of DNase I stora...

Страница 17: ...geneous distribution of the KingFisher Magnetic Beads in the container is essential before the beads are transferred to the wells or tubes in order to ensure a high consistency between the wells or tubes To gain complete resuspension of the beads shake the container vigorously or vortex briefly Avoiding Ribonuclease RNase Contamination RNA purity and integrity is essential for downstream applicati...

Страница 18: ...h use Redissolve precipitates by warming the solution at 37 C and equilibrate to room temperature 15 25 C Blood samples of certain species e g mouse contain a high amount of RNA With such species it is recommended to use up to 50 µl of blood sample to obtain RNA of equal yield and purity When working with less than a 200 µl volume of blood sample it is recommended to equalize the volume by adding ...

Страница 19: ...otocol will start 5 When the KingFisher Flex is to be run as a standalone instrument transfer the PURE_RNABlood_Flex96 protocol to the KingFisher Flex The instructions for transferring the protocol can be found in Chapter 4 Using the software in the BindIt Software for KingFisher Instruments version 3 2 User Manual 6 Add the KingFisher Magnetic Beads and isopropanol to the Sample plate during the ...

Страница 20: ...e purified RNA at 20 C or 80 C NOTE The final RNA concentration in the nuclease free water may increase if the purified RNA is eluted into a smaller than recommended volume of water but this can slightly reduce the overall RNA yield Instructions for KingFisher Duo with 12 pin Magnet Head These instructions are intended for RNA purification from 200 µl of blood using the KingFisher Pure RNA Blood K...

Страница 21: ...00 µl 20 µl 3 Fill the KingFisher Duo elution strip as follows Make sure that the elution strip is placed in the correct direction into the elution block Ensure that the perforated end is facing towards the user and the nuclease free water is pipetted into the correct wells Elution strip Content Reagent volume per well KingFisher Duo elution strip Nuclease free water 75 µl 4 Place a Thermo Scienti...

Страница 22: ...col run remove the plate from the instrument and add the KingFisher Magnetic Beads and isopropanol to row C on the Blood RNA plate Row Row name Add Added reagent volume per well C Sample Isopropanol KingFisher Magnetic Beads 400 µl 15 µl Resuspend the KingFisher Magnetic Beads well by vortexing before use 7 Place the plate back into the instrument and press OK 8 Add the Rebinding Buffer supplement...

Страница 23: ...tructions 2 If the PAXgene samples were stored in the cold equilibrate the samples at room temperature before purification according to the manufacturer s instructions for approx 2 h 3 Centrifuge the PAXgene blood RNA tubes for 10 min at 3 000 5 000 x g 4 Carefully discard the supernatant from each tube and wash the pellet Add 1 3 ml of nuclease free water vortex for 20 30 s and collect the pellet...

Страница 24: ... contents 5 Centrifuge the appropriate volume 1 2 ml of Tempus blood lysate in conical tubes at maximal speed about 12 000 16 000 g for 10 min at 4 C If the Tempus blood volume is more than 2 ml increase the time of centrifugation up to 30 min 6 Carefully discard the supernatant from each tube and add 450 µl of Lysis Buffer supplemented with DTT and 20 µl of Proteinase K Vortex the samples and inc...

Страница 25: ...d that A320 correction is used for the absorbance values Subtract the A320 from the A260 and A280 ratios to remove the effects of carryover of the magnetic particles Concentration of RNA sample 40 μg ml x A260 A320 x dilution factor Total amount of RNA isolated concentration x volume of sample in ml Purity of RNA sample A260 A320 A280 A320 ...

Страница 26: ......

Страница 27: ... the respective wells or tubes The amount of beads in the wells or tubes affects the yield of the purified RNA Binding Wash and Elution Steps The binding between the purified RNA and the KingFisher Magnetic Beads is strong in the presence of a chaotropic salt The volume of the Elution Buffer can be modified depending on user requirements concerning the purified RNA concentration The final RNA conc...

Страница 28: ...ize the yield of purified RNA avoid the lowest permitted volumes of Elution Buffer in the KingFisher instruments The Elution Buffer should cover the KingFisher Magnetic Beads completely and any possible sedimented magnetic bead pellet s should be completely resuspended In addition the volume of the Elution Buffer should be adequate for efficient mixing of the beads in order to obtain a maximal rel...

Страница 29: ...Magnetic Beads dry as this may result in lower elution efficiency Prolonged storage of the sample material may reduce the total RNA yield Use only Thermo Scientific plates strips and tip combs with the KingFisher instruments Use of products from other manufacturers may cause unsuitable mixing and affect the yield of purified RNA Low purity Prolonged storage of the sample material may reduce the qu...

Страница 30: ... the purification process Improper lysis may also cause problems collecting the KingFisher Magnetic Beads If the KingFisher Magnetic Beads are inefficiently collected from the Elution Buffer the addition of a small amount of detergent e g Tween 20 may improve the results KingFisher Magnetic Beads that occasionally remain attached to the tip combs at the end of the process do not affect the total R...

Страница 31: ...blood of different species using KingFisher magnetic particle processors Source Yield of RNA µg ml blood 1 Fresh human blood 11 20 2 Frozen human blood 7 17 3 Stabilized PAXgene Tempus human blood 9 12 4 Human bone marrow 15 80 5 Fresh mouse blood 120 160 6 Fresh rabbit blood 25 30 7 Fresh canine blood 11 15 8 Fresh animal horse cow sheep goat blood 2 5 ...

Страница 32: ......

Страница 33: ...er Flex 96 tip comb for PCR magnet 80 pcs 97002524 KingFisher Flex 96 tip comb for KF magnet 100 pcs 97002534 KingFisher Flex 96 tip comb for deep well magnet 100 pcs 97002610 KingFisher Flex 24 deep well tip comb and plate 50 pcs 97002540 KingFisher Flex 96 KF plate 200 µl 48 pcs 95040450 Microtiter deep well 96 plate 50 pcs 95040460 Microtiter deep well 96 plate sterile 50 pcs 95040470 KingFishe...

Страница 34: ...510 KingFisher Duo 6 tip comb for KingFisher Flex 24 deep well plate 48 pcs 97003520 KingFisher Duo elution strip 40 pcs 95040450 Microtiter deep well 96 plate 50 pcs 95040460 Microtiter deep well 96 plate sterile 50 pcs 95040470 KingFisher Flex 24 deep well plate 50 pcs 95040480 KingFisher Flex 24 deep well plate sterile 50 pcs 97003530 KingFisher Duo Combi pack for Microtiter deep well 96 plate ...

Страница 35: ...Thermo Scientific KingFisher Pure RNA Blood Kit 35 Thermo Fisher Scientific General Information Chapter C ...

Страница 36: ...ks are the property of Thermo Fisher Scientific Inc 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 Thermo Fisher Scientific 81 Wyman Street Waltham MA 02451 ...

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