Instructions for Use
Life Science Kits & Assays
Smart Bacteria DNA prep (a)
Order No.:
845-ASS-5508016 16 reactions
845-ASS-5508096 96 reactions
845-ASP-5508016 16 reactions
845-ASP-5508096 96 reactions
845-ASP-5508480 5x96 reactions
Publication No.: HB_ASP-5508_e_141016
This documentation describes the state at the time of publishing.
It needs not necessarily agree with future versions. Subject to change!
Print-out and further use permitted with indication of source.
© Copyright 2016, Analytik Jena AG, AJ Innuscreen GmbH
Manufacturer:
AJ Innuscreen GmbH
Robert-Rössle-Straße 10
13125 Berlin
Made in Germany!
Distribution/Publisher:
Analytik Jena AG
Konrad-Zuse-Straße 1
07745 Jena · Germany
Phone +49 3641 77 9400
Fax
+49 3641 77 767776
www.analytik-jena.com
[email protected]
Introduction
Contents
1
Introduction ........................................................................................... 3
1.1
Intended use ................................................................................ 3
1.2
Notes on the use of this manual ................................................. 4
2
Safety precautions ................................................................................. 5
3
Storage conditions ................................................................................ 6
4
Function testing and technical assistance ............................................ 7
5
Product use and warranty ..................................................................... 7
6
Kit components ..................................................................................... 8
6.1
Components not included in the kit......................................... 10
7
Recommended steps before starting ................................................ 10
8
GHS Classification .............................................................................. 11
9
8.1
Hazard phrases ......................................................................... 12
8.2
Precaution phrases ................................................................... 13
Product specifications ........................................................................ 14
10 Lysis of bacterial cells ......................................................................... 15
10.1 Isolation from liquid cultures ................................................... 15
10.2 Isolation of colonies from agar plates ..................................... 16
11 Preparation of Reagent Plates or Reagent Strips ............................. 17
11.1 General filling scheme.............................................................. 17
11.2 Piercing of sealing foil .............................................................. 17
12 Automated extraction using InnuPure® C16 .................................... 20
12.1 Sample tray of InnuPure® C16................................................. 20
12.2 Preparing sample tray of InnuPure® C16 ................................ 21
12.3 Handling of SmartExtraction Pipette Tips ............................... 23
12.4 Loading the sample to InnuPure® C16.................................... 24
12.5 Automatic processing of the InnuPure® C16 .......................... 24
13 Automated extraction using GeneTheatre ........................................ 27
13.1 Accessories needed .................................................................. 27
Smart Bacteria DNA prep (a)
Issue 10 / 2016
1
Introduction
13.2 Import of tube and plate data .................................................. 27
13.3 Preparing GeneTheatre............................................................ 28
13.4 Handling of SmartExtraction Tip ............................................. 30
13.5 Loading the sample to GeneTheatre ....................................... 30
13.6 Automatic processing of GeneTheatre .................................... 31
14 Semi-automated extraction using CyBio® SELMA 96 / 1000 μl ..... 32
14.1 Preparing CyBio® SELMA 96 / 1000 μl................................... 32
14.2 Handling of Smart Modified Pipette Tips ................................ 32
14.3 Loading the sample to CyBio® SELMA .................................... 33
14.4 Semi-automatic processing of CyBio® SELMA ........................ 34
15 Automated extraction using CyBio® FeliX......................................... 35
15.1 Accessories needed .................................................................. 35
15.2 Preparing CyBio® FeliX ............................................................ 35
15.3 Handling of SmartExtraction Tips ........................................... 36
15.4 Loading the sample to CyBio® FeliX ........................................ 37
15.5 Automatic processing of CyBio® FeliX ..................................... 37
16 Troubleshooting ................................................................................. 38
17 Related Products ................................................................................ 39
2
Smart Bacteria DNA prep (a)
Issue 10 / 2016
Introduction
1
Introduction
1.1
Intended use
The smart Bacteria DNA prep (a) has been designed for automated isolation of high molecular weight genomic DNA (gDNA) from bacteria cells
derived from liquid cultures or agar plates. The kit utilizes the new
SmartExtraction technology invented by Analytik Jena (patent pending).
The procedure starts with the pelleting of bacterial cells from liquid cultures or with the picking colonies from an agar plate. Following lysis of
bacteria cells, samples are transferred into the Reagent Strips or Reagent
Plate of the kit, which are already prefilled with all extraction reagents
needed for the automated isolation process using a unique 1 mL filter tip.
The extraction process is based on adsorption of the genomic DNA to
Smart Modified Surfaces inside the tip. After washing the genomic DNA is
eluted from the Smart Modified Surfaces and is ready for use in subsequent downstream applications.
The whole extraction process just needs simple pipetting up and down.
The combination of patented, low-salt DC-Technology® with patentpending Smart Modified Surface is optimized to get a maximum of yield
and quality.
CONSULT INSTRUCTION FOR USE
This package insert must be read carefully prior to use. Package insert instructions must be followed accordingly. Reliability of results cannot be
guaranteed if there are any deviations from the instructions in this package insert.
Smart Bacteria DNA prep (a)
Issue 10 / 2016
3
Introduction
1.2
Notes on the use of this manual
For easy reference and orientation, the manual uses the following warning and information symbols as well as the shown methodology:
Symbol
Information
REF
Catalogue number
N
Content
Contains sufficient reagents for <N> tests
Storage conditions
Store at room temperature or shown conditions respectively
Consult instructions for use
This information must be observed to avoid improper use of the kit
and the kit components.
Used by
Expiry date
Lot number
The number of the kit charge
Manufactured by
Contact information of manufacturer
For single use only
Do not use components for a second time

Note / Attention
Observe the notes marked in this way to ensure correct function of
the device and to avoid operating errors for obtaining correct results.
The following systematic approach is introduced in the manual:
4

The chapters and figures are numbered consecutively.

A cross reference is indicated with an arrow (e.g.  Notes on the use
of this manual" p. 4).

Working steps are numbered.
Smart Bacteria DNA prep (a)
Issue 10 / 2016
Safety precautions
2
Safety precautions
NOTE
Read through this chapter carefully prior to guarantee your own safety
and a trouble-free operation.
Follow all the safety instructions explained in the manual, as well as all
messages and information, which are shown.
All due care and attention should be exercised in handling the materials
and reagents contained in the kit. Always wear gloves while handling
these reagents and avoid any skin contact! In case of contact, flush eyes
or skin with a large amount of water immediately.
FOR SINGLE USE ONLY!
This kit is made for single use only!
ATTENTION!
Don’t eat or drink components of the kit!
The kit shall only be handled by educated personal in a laboratory environment!
If the buffer bottles are damaged or leaking, wear gloves and protective
goggles when discarding the bottles in order to avoid any injuries. Analytik Jena AG has not tested the liquid waste generated during using the kit
for potential residual infectious components. This case is highly unlikely,
but cannot be excluded completely. Therefore, all liquid waste must be
considered as potentially infectious and must be handled and discarded
according to local safety regulation.
Please observe the federal, state and local safety and environmental
regulations. Observe the usual precautions for applications using extracted nucleic acids. All materials and reagents used for DNA or RNA isolation
should be free of DNases or RNases.
Smart Bacteria DNA prep (a)
Issue 10 / 2016
5
Storage conditions
ATTENTION!
Do not add bleach or acidic components to the waste after sample preparation!
NOTE
Emergency medical information in English and German can be obtained 24
hours a day from:
Poison Information Center, Freiburg / Germany
Phone: +49 (0)761 19 240.
For more information, please ask for the material safety data sheets
(MSDS’s).
3
Storage conditions
The smart Bacteria DNA prep (a) should be stored dry at room temperature (15 °C to 30 °C). When stored at room temperature, the kit is stable
until the expiration date printed on the label on the kit box. The enzymes
inside should be stored at 4 °C to 8 °C or at -22 °C to -18 °C. Please follow
the instructions printed below in table Kit components (→ p. 8).
If there are any precipitates within the provided solutions solve these precipitates by careful warming. Or take care not to carry them over into the
sample preparation procedure. Before every use make sure that all components have room temperature. For further information see table Kit
components (→ p. 8).
6
Smart Bacteria DNA prep (a)
Issue 10 / 2016
Function testing and technical assistance
4
Function testing and technical assistance
The Analytik Jena AG guarantees the correct function of the kit for applications as described in the manual. Each lot of smart Bacteria DNA prep
(a) is tested against predetermined specifications to ensure consistent
product quality in accordance with the ISO-certified Quality Management
System of Analytik Jena AG.
We reserve the right to change or modify our products to enhance their
performance and design. If you have any questions or problems regarding
any aspects of the smart Bacteria DNA prep or other Analytik Jena AG
products, please do not hesitate to contact us. For technical support or
further information in Germany please dial +49 36 41 / 77 94 00. For
other countries please contact your local distributor.
5
Product use and warranty
The user is responsible to validate the performance of the Analytik Jena
AG kits for any particular use, since the performance characteristics of our
kits have not been validated for any specific application. Analytik Jena AG
kits may be used in clinical diagnostic laboratory systems after the laboratory has validated the complete diagnostic system as required by CLIA’ 88
regulations in the U.S. or equivalents in other countries.
All products sold by the Analytik Jena AG are subjected to extensive quality control procedures and are warranted to perform as described when
used correctly. Any problems should be reported immediately.
NOTE
For research use only!
Smart Bacteria DNA prep (a)
Issue 10 / 2016
7
Kit components
6
Kit components
IMPORTANT
Store lyophilized Proteinase K at 4 ° to 8 °C. Divide dissolved Proteinase
K into aliquots and storage at -22 °C to -18 °C is recommended. Repeated freezing and thawing will reduce the activity dramatically!
Store lyophilized and dissolved Enzyme A and Enzyme B at -22 °C to 18 °C.
Store the Enzyme C at -22 to -18 °C.
STORAGE CONDITIONS
All other components are stored at room temperature.
16
8
96
480
845-AS[S/P]5508016
845-AS[S/P]5508096
5x 845-AS[S/P]5508096
SmartExtraction
Tips
16
96
5x 96
Proteinase K
for 1 x 1.5 ml
working solution
for 4 x 1.5 ml
working solution
for 20 x 1.5 ml
working solution
Lysis Solution CBV
5 ml
30 ml
5 x 30 ml
Enzyme A
1
5
5x5
Storage Buffer A
1 x 270 μl
5 x 270 μl
25 x 270 μl
Enzyme B
1
5
5x5
Storage Buffer B
1 x 150 μl
5 x 150 μl
25 x 150 μl
Enzyme C
1 x 125 μl
5 x 125 μl
25 x 125 μl
Binding Optimizer
1 ml
5 ml
5 x 5 ml
TE-Buffer
5 ml
20 ml
5 x 20 ml
Reagent Strips L*
(* Depending on
order)
16
(pre-filled, sealed)
96
(pre-filled, sealed)
5 x 96
(pre-filled, sealed)
Reagent Plates L*
2
12
5 x 12
Smart Bacteria DNA prep (a)
Issue 10 / 2016
Kit components
(* Depending of order)
(pre-filled, sealed)
(pre-filled, sealed)
(pre-filled, sealed)
Filter Tips
1 x 16
1 x 96
5 x 96
Elution Tubes
(0.65 ml)
16
2 x 48
10 x 48
Elution Caps
(Stripes)
2
12
5 x 12
Manual
1
1
1
16
Initial steps
Smart Bacteria DNA prep (a)
Issue 10 / 2016
96
480
845-AS[S/P]5508016
845-AS[S/P]5508096
5x 845-AS[S/P]5508096
Proteinase K
Dissolve by addition of 1.5 ml of
ddH2O, mix thoroughly and store
as described
above.
Proteinase K
Dissolve by addition of 1.5 ml of
ddH2O, mix thoroughly and store
as described
above.
Proteinase K
Dissolve by addition of 1.5 ml of
ddH2O, mix thoroughly and store
as described
above.
Enzyme A
Dissolve by addition of 250 μl of
Storage Buffer A,
mix thoroughly
and store as described above.
Enzyme A
Dissolve by addition of 250 μl of
Storage Buffer A,
mix thoroughly
and store as described above.
Enzyme A
Dissolve by addition of 250 μl of
Storage Buffer A,
mix thoroughly
and store as described above.
Enzyme B
Dissolve by addition of 125 μl of
Storage Buffer B,
mix thoroughly
and store as described above.
Enzyme B
Dissolve by addition of 125 μl of
Storage Buffer B,
mix thoroughly
and store as described above.
Enzyme B
Dissolve by addition of 125 μl of
Storage Buffer B,
mix thoroughly
and store as described above.
9
Recommended steps before starting
6.1
7
Components not included in the kit

15 ml tubes

2 ml tubes

ddH2O for dissolving Proteinase K
Recommended steps before starting

Heat thermal mixer (shaking platform) at 37 °C.

Ensure that Proteinase K, Enzyme A and Enzyme B has prepared according to the instructions ( “Kit components” p. 8).

Ensure that Proteinase K, Enzyme A and Enzyme B has prepared according to the instructions ( "Kit components" p. 8).

Prepare the Enzyme Mix according table printed below.
Amount of reactions
Enzyme A
Enzyme B
Enzyme C
1x
12 μl
7 μl
7 μl
2x
24 μl
14 μl
14 μl
3x
36 μl
21 μl
21 μl
16 x
180 μl
90 μl
90 μl
X reactions
12 x X μl
7 x X μl
7 x X μl
Store prepared Enzyme Mix at -22 °C to -18 °C. The prepared Enzyme Mix
is stable for 21 days at -22 °C to -18 °C. After that time you have to prepare it fresh.
10
Smart Bacteria DNA prep (a)
Issue 10 / 2016
GHS Classification
8
GHS Classification
Component
Hazard
contents
Binding
Optimizer
Acetic acid
10–≤25 %
GHS Symbol
Hazard
phrases
Precaution
phrases
315, 319
101, 102, 103,
280,
305+351+338,
362, 302+352,
403+P233, 501
225, 319,
336
101, 102, 103,
210, 233, 240,
305+351+338,
403+235
225, 319
101, 102, 103,
210, 240,
305+351+338,
403+235
315, 319,
334, 317,
335
101, 102, 103,
261, 280,
305+351+338,
342+311, 405,
501
Warning
Propan-2-ol
Propan-2-ol
50–100 %
Danger
Ethanol
Ethanol
50–100 %
Danger
Proteinase K
Smart Bacteria DNA prep (a)
Proteinase,
Tritirachium
album serine
Issue 10 / 2016
Danger
11
GHS Classification
8.1
12
Hazard phrases
225
Highly flammable liquid and vapor.
315
Causes skin irritation.
317
May cause an allergic skin reaction.
319
Causes serious eye irritation.
334
May cause allergy or asthma symptoms or breathing difficulties if
inhaled.
335
May cause respiratory irritation.
336
May cause drowsiness or dizziness.
Smart Bacteria DNA prep (a)
Issue 10 / 2016
GHS Classification
8.2
Precaution phrases
101
If medical advice is needed, have product container or label at
hand.
102
Keep out of reach of children.
103
Read label before use.
210
Keep away from heat, hot surfaces, sparks, open flames and
other ignition sources. No smoking.
233
Keep container tightly closed.
240
Ground/bond container and receiving equipment.
261
Avoid breathing dust/fume/gas/mist/vapors/spray.
264
Wash thoroughly after handling.
280
Wear protective gloves/protective clothing/ eye protection/face protection.
362
Take off contaminated clothing.
403
Store in well-ventilated place.
405
Store locked up.
501
Dispose of contents/container in accordance with local/regional/national/international regulations.
302+352
IF ON SKIN: Wash with plenty of water.
337+313
If eye irritation persists: Get medical advice/attention.
342+311
If experiencing respiratory symptoms: Call a POISON
CENTER/doctor.
403+233
Store in a well-ventilated place. Keep container tightly closed.
403+235
Store in a well-ventilated place. Keep cool.
305+351+ IF IN EYES: Rinse cautiously with water for several minutes.
338
Remove contact lenses, if present and easy to do. Continue
rinsing.
Smart Bacteria DNA prep (a)
Issue 10 / 2016
13
Product specifications
9
Product specifications
1.
2.
Starting material:

Up to 2 x 109 of bacterial cells

Gram- or gram+
Time for isolation:

Lysis of bacterial cell walls: approx. 30 minutes

Proteolyse:
approx. 30 minutes

Standard protocol:
approx. 32 minutes (InnuPure® C16)

Fast protocol:
approx. 21 minutes (InnuPure® C16)

Sensitive protocol:
approx. 67 minutes (InnuPure® C16)
NOTE
For samples with a low or unknown content of DNA always use „Standard
protocol” or “Sensitive protocol” for maximum yield. The “fast protocol” is
only recommended for samples with high DNA content in combination
with time-critical applications.
3.
14
Typical yield:

Depending on amount of starting material and condition of bacterial cells

Yield of isolated DNA is affected by the concentration of starting
material. An over-night-culture has of course another cell concentration and, in the end, another yield of isolated DNA as
working with a colony picked up from an agar plate.
Smart Bacteria DNA prep (a)
Issue 10 / 2016
Lysis of bacterial cells
10
Lysis of bacterial cells
NOTE
The kit has been optimized for isolation of genomic DNA from bacterial
cultures up to 2 x 109 cells.
10.1
Isolation from liquid cultures
1.
Transfer the bacterial culture (volume depends on the concentration
of starting material) into a tube, for example 2 ml of a 1 x 109 culture into a 2 ml tube.
2.
Centrifuge to pellet the culture (e.g. 10 minutes at 3,000 x g).
3.
Carefully discard the supernatant by pipetting. Do not discard the
pellet!
4.
Add 170 μl TE-Buffer and resuspend the pellet carefully.
5.
Vortex the Enzyme Mix (prepared as described on p.7) thoroughly.
6.
Add 20 μl of the Enzyme Mix. Vortex the sample shortly.
7.
Incubate sample for 30 minutes at 37 °C and 550 rpm in a shaking
platform.
8.
After finishing this step preheat the shaking platform at 50 °C.
9.
Add 200 μl Lysis Solution CBV and 30 μl Proteinase K to sample.
10. Vortex the sample shortly.
11. Incubate sample for 30 minutes at 50 °C and 550 rpm in a shaking
platform.
Proceed with “Preparation of Reagent Plates or Reagent Strips” on p. 17.
NOTE
The lysed sample will be processed using a liquid handling platform.
Please follow the instruction of the manual from chapter “Preparation of
Reagent Plates or Reagent Strips” on p. 17.!
Smart Bacteria DNA prep (a)
Issue 10 / 2016
15
Lysis of bacterial cells
10.2
Isolation of colonies from agar plates
1.
Add 170 μl TE-Buffer to a 2.0 ml tube.
2.
Pick up the colonies from the plate with a sterile inoculation loop.
3.
Transfer the colonies to the tube with TE-Buffer.
4.
Vortex the prepared Enzyme Mix thoroughly.
5.
Add 20 μl of the Enzyme Mix. Vortex the sample shortly.
6.
Incubate sample for 30 minutes at 37°C and 550 rpm in a shaking
platform.
7.
After finishing this step, preheat the shaking platform to 50°C.
8.
Add 200 μl Lysis Solution CBV and 30 μl Proteinase K to the sample.
9.
Vortex the sample shortly
10. Incubate sample for 30 minutes at 50°C and 550 rpm in a shaking
platform.
Proceed with “Preparation of Reagent Plates or Reagent Strips” on p. 17.
NOTE
The lysed sample will be processed using a liquid handling platform.
Please follow the instruction of the manual from chapter “Preparation of
Reagent Plates or Reagent Strips” on p. 17.!
16
Smart Bacteria DNA prep (a)
Issue 10 / 2016
Preparation of Reagent Plates or Reagent Strips
11
Preparation of Reagent Plates or Reagent Strips
11.1
General filling scheme
11.2
Cavity 1:
Empty
Cavity 7:
Washing Solution
Cavity 2:
Empty
Cavity 8:
Empty
Cavity 3:
Empty
Cavity 9:
Elution Buffer
Cavity 4:
Binding Solution
Cavity 10:
Empty
Cavity 5:
Washing Solution
Cavity 11:
Empty
Cavity 6:
Washing Solution
Cavity 12:
Empty
Piercing of sealing foil
NOTE
Invert the reagent strips or reagent plates for 3 – 4 times and thump it
onto a table to collect the pre-filled solutions at the bottom of the single
strips or reagent plates.
Smart Bacteria DNA prep (a)
Issue 10 / 2016
17
Preparation of Reagent Plates or Reagent Strips
Reagent Plates and Reagent Strips are
prefilled with extraction reagents and are
sealed with a foil. Prior the usage this foil
has to be pierced manually, by using the
piercing tools (single piercer or 8fold
piercer).
Hold the reagent plates or strips horizontal to avoid spilling of the reagents while
piercing of the foil.
Open all cavities (one row per sample).
Using 8 samples in parallel
Using single samples
18
Smart Bacteria DNA prep (a)
Issue 10 / 2016
Preparation of Reagent Plates or Reagent Strips
NOTE!
The lysed sample will be processed using a liquid handling platform.
Please follow the instruction of the manual from the following chapters:
Automated extraction using InnuPure® C16
Automated extraction using GeneTheatre
Semi-automated extraction using CyBio® SELMA 96 / 1000 μl
Automated extraction using CyBio® FeliX
on p. 20
on p. 27
on p. 32
on p. 35
Pay special attention to sub-chapter “Loading the sample”.
Smart Bacteria DNA prep (a)
Issue 10 / 2016
19
Automated extraction using InnuPure® C16
12
Automated extraction using InnuPure® C16
12.1
Sample tray of InnuPure® C16
20
No. 1:
SmartExtraction and standard filter tips
No. 2:
Elution vessels for purified samples
No. 3:
Tip block
No. 4:
Pressure pad
No. 5:
Sample block for reagent plates or adapter for reagent
strips
No. 6:
Serrated guide rail
No. 7:
Adapter for reagent strips
Smart Bacteria DNA prep (a)
Issue 10 / 2016
Automated extraction using InnuPure® C16
12.2
Preparing sample tray of InnuPure® C16
NOTE
The needed number of reagent strips or reagent plates is depending on
the number of samples, which have to be processed. Don't use more strips
as number of samples!
1.
Move the InnuPure® C16 sample tray into the priming station and
fold the holding-down clamp at the sample tray upwards!
2.
Place the reagent plate or an adapter for the reagent strips into the
holder of the sample tray. Here the colored marking on the adapter
must point to the colored point at the holder.
Reagent Plate
The flattened corners of the reagent
plate must point to the colored dot on the
holder.
Reagent Strips
Place the strips into the adapter. The
long tab marked with the label "AJ" must
point to the engraved numbers on the
sample tray.
AJ
CAUTION
Both holders have to be equipped with a plate or strips. If applicable use
an empty or already used plate for the respecting holder.
Smart Bacteria DNA prep (a)
Issue 10 / 2016
21
Automated extraction using InnuPure® C16
3.
Fold down the holding-down clamp to prevent the plates and strips
to be pulled out of the holder during the extraction process.
4.
Place the Elution Tubes into the wider drill hole at the edge of the tip
block. Empty sample positions do not need to be filled.
NOTE
Especially with the strips make sure that for every strip the tips and the
elution vessel are in the corresponding positions in the tip block!
5.
For correct placing the tips refer to chapter “Handling of SmartExtraction Pipette Tips” on p. 23!
IMPORTANT NOTE
Use Elution Tubes (0.65 ml) with corresponding Elution Caps.
22
Smart Bacteria DNA prep (a)
Issue 10 / 2016
Automated extraction using InnuPure® C16
12.3
Handling of SmartExtraction Pipette Tips
 
Sample
Tray
Checking the SmartExtraction tips.
Make sure that the Smart Modified Material
is collected near the outlet of the Pipette Tip.
If necessary flip the Pipette Tip by finger or
edge of table or invert the Pipette Tip a few
times. The optimal position of the Smart
Modified Material inside the Pipette Tip is
shown in the picture on the left side.
Loading Pipette Tips to InnuPure® C16.
The SmartExtraction Tips are inserted in the
tip row 1. The tip row 1 is the tip row adjacent to the reagent plates or strips. See figure
left.
(Top View)
Tip Block
Smart Bacteria DNA prep (a)
Issue 10 / 2016
Tip row 1 (SmartExtraction Tip)
Tip row 2 (standard filter tips)
Elution Tubes
23
Automated extraction using InnuPure® C16
12.4
Loading the sample to InnuPure® C16
NOTE
The following step will be done after the sample lysis!
12.5
1.
Transfer the whole sample into the first cavity (closed to the graved
numbers of the sample tray) of Reagent Strips L or Reagent Plates L.
2.
Transfer 40 μl of Binding Optimizer to the lysed sample into the first
cavity of Reagent Strips L or Reagent Plates L.
Automatic processing of the InnuPure® C16
1.
Switch on the InnuPure® C16 and wait for the device initialization to
complete, which is signaled by a beeping sound.
2.
Move the loaded sample tray with the reagent strips forward into the
adapter on the front of the InnuPure® C16. The serrated rails at the
side of the sample tray must protrude into the grooves of the adapter. After pressing lightly against the tip block the sample tray is automatically pulled into the device.
IMPORTANT – CAUTION
Risk of crushing
Immediately let go of the sample tray once it is being pulled
in. Otherwise there is a risk of
your hand being crushed.
24
Smart Bacteria DNA prep (a)
Issue 10 / 2016
Automated extraction using InnuPure® C16
3.
Start the extraction protocol:

Press [SELECT PROTOCOL] in the starting window.

Select the desired extraction protocol
“SE_Ext_Lysis_C16_01”or
“SE_Ext_Lysis_Fast_C16_01” or
“SE_Ext_Lysis_Sensitive_C16_01” and press [START].

Enter elution volume and press [OK].
Starting material
Recommended elution volume
Colonies
min. 200 μl
< 1 x 108
min. 200 μl
> 1 x 108
min. 300 μl
4.
If needed, choose log-file and enter sample-ID’s, press [OK] or
[CANCEL].
NOTE
It is possible to enter sample-ID‘s and to create a run log-file. Find more
detailed information how to start an extraction protocol using InnuPure®
C16 inside the user manual “6.3.5 Using the sample setup tool” on page
37!
5.
After completion of the protocol press [NEXT] and the sample tray is
then automatically moved out of the device.
NOTE
The chosen protocol is performed by the device and after the protocol is
finished, the tray with the purified samples will be moved out after press
[NEXT] and the message 'Program finished' is shown in the screen of the
device!
Smart Bacteria DNA prep (a)
Issue 10 / 2016
25
Automated extraction using InnuPure® C16
6.
Remove the sample tray from the adapter of the InnuPure® C16 and
move it back into the priming station.
7.
After finishing the extraction protocol, the Elution Tubes (0.65 ml)
on position 2 contain the extracted DNA; close the lids and store the
DNA under proper conditions.
NOTE
Be careful when closing and
store the elution vessels.
NOTE
Store the DNA under adequate conditions. We recommend storing the extracted DNA at -22 to -18 °C!
26
Smart Bacteria DNA prep (a)
Issue 10 / 2016
Automated extraction using GeneTheatre
13
Automated extraction using GeneTheatre
13.1
Accessories needed
13.2

8 channel pipette head 1000 μl (844-00415-0) or optional 1 channel pipette head 1000 μl (844-00414-0)

Tip adapter 96 / 1000 μl (OL3317-11-140)

Optional: Adapter for Reagent Strips (845-60006-0)

Waste Box for used tips (844-00430-0)

Height Adapter 40 mm (844-00445-0)
Import of tube and plate data
1.
Copy "Components.gttub" and "Components.gtplt" to an USB memory
stick.
2.
Switch on GeneTheatre and open GeneTheatre software.
3.
Select menu command FILE / IMPORT / IMPORT CAVITIES and click
on OPEN.
4.
Select cavity file "Components.gttub" from USB memory stick to import data to GeneTheatre software.
5.
Select the required tubes/cavities and click PASTE.
NOTE
Multiple selections are possible with the shift or Ctrl key pressed.
6.
Select menu command FILE / IMPORT / IMPORT CAVITIES and click
on OPEN.
7.
Select cavity file "Components.gtplt" from USB memory stick to import data to GeneTheatre software.
8.
Select the required plate data and click PASTE.
Smart Bacteria DNA prep (a)
Issue 10 / 2016
27
Automated extraction using GeneTheatre
NOTE
Multiple selections are possible with the shift or Ctrl key pressed.
13.3
Preparing GeneTheatre
NOTE
The needed number of reagent strips or reagent plates is depending on
the number of samples, which have to be processed. Don't use more strips
as number of samples!
Invert the reagent strips or reagent plates for 3 – 4 times and thump it
onto a table to collect the pre-filled solutions at the bottom of the single
strips or reagent plates.
1.
Place the empty waste box onto deck position C1.
2.
Place the 40 mm height adapter onto deck position D2.
3.
Place one opened Reagent Plate or optional one adapter with Reagent Strips onto the 40 mm height adapter (deck position D2).
NOTE
For Reagent Plates the flattened corners of the reagent plate must be orientated to the upper corners of deck position D2. For Reagent Plates, the
AJ label must be orientated to left side of deck position D2.
28
Smart Bacteria DNA prep (a)
Issue 10 / 2016
Automated extraction using GeneTheatre
AJ
Deck layout using
Reagent Plate
Deck layout using Reagent Strips
4.
Place the Tip adapter 96 / 1000 μl onto deck position D1.
5.
Place one SmartExtraction Tip per sample to the column 1 of the Tip
adapter.
NOTE
Especially with the strips make sure that for every strip the tips are in the
corresponding positions in the tip adapter!
Smart Bacteria DNA prep (a)
Issue 10 / 2016
29
Automated extraction using GeneTheatre
13.4
Handling of SmartExtraction Tip
 
13.5
Checking the SmartExtraction tips.
Make sure that the Smart Modified Material is collected near the outlet of the Pipette Tip. If necessary
flip the Pipette Tip by finger or edge of table or invert the Pipette Tip a few times. The optimal position of the Smart Modified Material inside the Pipette Tip is shown in the picture on the left side.
Loading the sample to GeneTheatre
NOTE
The following step will be done after the sample lysis!
30
1.
Transfer the sample (max. 400 μl) into the first (left) cavity of Reagent Strips L or Reagent Plates L.
2.
Transfer 40 μl of Binding Optimizer to the lysed sample into the first
cavity of Reagent Strips L or Reagent Plates L.
Smart Bacteria DNA prep (a)
Issue 10 / 2016
Automated extraction using GeneTheatre
13.6
Automatic processing of GeneTheatre
1.
Switch on the GeneTheatre and open GeneTheatre software.
2.
Load the extraction protocol:
“SE_Ext_Lysis_GT_01”or
“SE_Ext_Lysis_Fast_GT_01”
Open pipetting step Elute and adjust Elution Volume; press [OK].
Starting material
Recommended elution volume
Colonies
min. 200 μl
< 1 x 108 cells
min. 200 μl
> 1 x 108 cells
min. 300 μl
3.
After finishing the extraction protocol, the cavity 12 of the reagent
plastics contains the extracted DNA. Store the DNA under proper
conditions.
NOTE
Store the DNA under adequate conditions. We recommend storing the extracted DNA at -22 to -18 °C!
Smart Bacteria DNA prep (a)
Issue 10 / 2016
31
Semi-automated extraction using CyBio® SELMA 96 / 1000 μl
14
Semi-automated extraction using CyBio® SELMA 96 /
1000 μl
14.1
Preparing CyBio® SELMA 96 / 1000 μl
1.
Install CyBio® SELMA tray in guide groove position II.
2.
Place the opened Reagent Plate to Working Position 2 (shiftable in
column-steps).
NOTE
The flattened corners of the reagent plate must be orientated to the upper corners of Working Position 2.
3.
14.2
Add up to 8 SmartExtraction Tips to a 1 ml tip tray (column 1) and
move it into the tip holder of CyBio® SELMA by using [Change Tips].
Handling of Smart Modified Pipette Tips
 
32
Checking the SmartExtraction tips.
Make sure that the Smart Modified Material is collected near the outlet of the Pipette Tip. If necessary
flip the Pipette Tip by finger or edge of table or invert the Pipette Tip a few times. The optimal position of the Smart Modified Material inside the Pipette Tip is shown in the picture on the left side.
Smart Bacteria DNA prep (a)
Issue 10 / 2016
Semi-automated extraction using CyBio® SELMA 96 / 1000 μl
14.3
Loading the sample to CyBio® SELMA
NOTE
The following step will be done after the sample lysis!
1.
Pipette the needed amount of Elution Volume from cavity 9 to cavity
12 manually.
Starting material
Recommended elution volume
Colonies
min. 200 μl
< 1 x 108
min. 200 μl
> 1 x 108
min. 300 μl
2.
Transfer the whole sample (max. 400 μl) into the first (left) cavity
Reagent Plates L.
3.
Transfer 40 μl of Binding Optimizer to the lysed sample into the first
cavity of Reagent Strips L or Reagent Plates L.
Smart Bacteria DNA prep (a)
Issue 10 / 2016
33
Semi-automated extraction using CyBio® SELMA 96 / 1000 μl
14.4
Semi-automatic processing of CyBio® SELMA
1.
Switch on the CyBio® SELMA and start initialization.
2.
Choose Pipetting and transfer 350 μl of Binding Buffer from cavity 4
to the sample in cavity 1.
3.
Choose Priming and follow the steps shown in the list below.
Step
Cavity
Mixing Volume
Cycles
Speed
Height
Binding
1
500 μl
150
550 μl/sec
68.5 mm
Washing 1
5
600 μl
10
200 μl/sec
68.5 mm
Washing 2
6
600 μl
10
200 μl/sec
68.5 mm
Washing 3
7
600 μl
10
200 μl/sec
68.5 mm
Drying
8
800 μl
100
550 μl/sec
62.5 mm
Elution
12
Choosen Elution
Volume minus
50 μl
150
400 μl/sec
72.0 mm
NOTE
Store the DNA under adequate conditions. We recommend storing the extracted DNA at -22 to -18 °C!
For further information on programming CyBIO® SELMA, please refer to
device user manual.
Steps can be stored as templates and re-used.
34
Smart Bacteria DNA prep (a)
Issue 10 / 2016
Automated extraction using CyBio® FeliX
15
Automated extraction using CyBio® FeliX
15.1
Accessories needed
15.2

8-channel CHOICE™ 10 μl – 1000 μl Adapter (OL3316-11-330)

Tip rack 96/1000 μl (OL3317-11-140)

Optional: Adapter for Reagent Strips (845-60006-0)

Waste Box I (small) (844-00430-0)
Preparing CyBio® FeliX
NOTE
The needed number Reagent Strips or Reagent Plates is depending on the
number of samples, which have to be processed. Don't use more strips as
number of samples!
Invert the reagent strips or reagent plates for 3 – 4 times and thump it
onto a table to collect the pre-filled solutions at the bottom of the single
strips or reagent plates.
AJ
Deck layout using
Reagent Plate
Smart Bacteria DNA prep (a)
Issue 10 / 2016
Deck layout using Reagent Strips
35
Automated extraction using CyBio® FeliX
1.
Place the empty waste box onto deck position A3.
2.
Place the 8-channel CHOICE™ 10 μl – 1000 μl Adapter onto deck
position A6.
3.
Place one opened Reagent Plate or optional one Adapter with Reagent Strips onto deck position A5.
NOTE
For Reagent Plates the flattened corners of the Reagent Plate must be
orientated to the upper corners of deck position A5. For Reagent Plates,
the AJ label must be orientated to left side of deck position A5.
4.
Place the Tip adapter 96 / 1000 μl onto deck position A4.
5.
Place one SmartExtraction Tip per sample to the column 1 of the Tip
adapter.
NOTE
Especially with the strips make sure that for every strip the tips are in the
corresponding positions in the tip adapter!
15.3
Handling of SmartExtraction Tips
 
36
Checking the SmartExtraction tips.
Make sure that the Smart Modified Material is collected near the outlet of the Pipette Tip. If necessary
flip the Pipette Tip by finger or edge of table or invert the Pipette Tip a few times. The optimal position of the Smart Modified Material inside the Pipette Tip is shown in the picture on the left side.
Smart Bacteria DNA prep (a)
Issue 10 / 2016
Automated extraction using CyBio® FeliX
15.4
Loading the sample to CyBio® FeliX
NOTE
The following step will be done after the sample lysis!
15.5
1.
Transfer the whole sample (max. 400 μl) into the first (left) cavity of
Reagent Strips L or Reagent
Plates L.
2.
Transfer 40 μl of Binding Optimizer to the lysed sample into the first
cavity of Reagent Strips L or Reagent Plates L.
Automatic processing of CyBio® FeliX
1.
Switch on the CyBio® FeliX and open Composer.
2.
Load the extraction protocol:
“SE_Ext_Lysis_FX_01”or
“SE_Ext_Lysis_Fast_FX_01”
Open pipetting step Elute and adjust Elution Volume; press [OK].
Starting material
Recommended elution volume
Colonies
min. 200 μl
< 1 x 108
min. 200 μl
> 1 x 108
min. 300 μl
3.
After finishing the extraction protocol, the cavity 12 of the reagent
plastics contains the extracted DNA. Store the DNA under proper
conditions.
NOTE
Store the DNA under adequate conditions. We recommend storing the extracted DNA at -22 to -18 °C!
Smart Bacteria DNA prep (a)
Issue 10 / 2016
37
Troubleshooting
16
Troubleshooting
Problem / probable cause
Comments and suggestions
Low amount of extracted DNA

Insufficient lysis
Increase lysis time.
Reduce amount of starting material.

Incomplete elution
Prolong the incubation time with
Elution Buffer or repeat elution
step once again (only CyBio®
SELMA).

Smart Modified Material not collected
near the tip opening
Flip the Pipette Tip by finger or
edge of table or invert the Pipette
Tip a few times to collect Granulates at the lower part of pipette
tip.

Preparation without Binding Optimizer
It is important to add the Binding
Optimizer to the Reagent Plastic as
described in chapters for handling
of the liquid handling platforms.
Binding Optimizer need to be added after lysis of sample is finished!
High viscosity extracted DNA

Insufficient amount of Elution Buffer
Elute the DNA with a higher volume of Elution Buffer.
Degraded or sheared DNA
38

Old material insufficient
Old material often contains degraded DNA.

RNA contaminations of extracted DNA
RNase A digestion
Smart Bacteria DNA prep (a)
Issue 10 / 2016
Related Products
17
Related Products
Name
Amount
Order No.
Additional products for nucleic acid purification
innuPREP Proteinase K
6 mg
845-CH-0010006
30 mg
845-CH-0010030
16 rxn (Strips)
845-ASS-1208016
96 rxn (Strips)
845-ASS-1208096
16 rxn (Plates)
845-ASP-1208016
96 rxn (Plates)
845-ASP-1208096
5x96 rxn (Plates)
845-ASP-1208480
16 rxn (Strips)
845-ASS-2008016
96 rxn (Strips)
845-ASS-2008096
16 rxn (Plates)
845-ASP-2008016
96 rxn (Plates)
845-ASP-2008096
5x96 rxn (Plates)
845-ASP-2008480
16 rxn (Strips)
845-ASS-5608016
96 rxn (Strips)
845-ASS-5608096
16 rxn (Plates)
845-ASP-5608016
96 rxn (Plates)
845-ASP-5608096
5x96 rxn (Plates)
845-ASP-5608480
Automated nucleic acid purification
smart Blood DNA Midi prep (a)
smart DNA prep (a)
Smart Yeast DNA prep (a)
Smart Bacteria DNA prep (a)
Issue 10 / 2016
39
engl. 10/16 – Analytik Jena AG, Jena
Analytik Jena AG
Konrad-Zuse-Str. 1
07745 Jena · Germany
Phone +49 3641 77 70
Fax +49 3641 77 9279
[email protected]
www.analytik-jena.com
Pictures: Analytik Jena AG
Subject to changes in design and scope of delivery as well as further technical development!
© Analytik Jena AG
Headquarters