HumaCycler

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Cat No. 86100/5

REVISION LIST OF THE MANUAL
Rev. /DATE. REVISION DESCRIPTION
01/2015-05 First edition

SYSTEM VERSION

COPYRIGHT

Copyright 2015, Human Gesellschaft fr Biochemica und Diagnostica mbH, Wiesbaden,

Germany. All rights reserved.

No part of this documentation may be reproduced in any form, nor processed, copied or
distributed by means of electronic systems, without prior permission of HUMAN in writ-
ing. Since all precautionary measures were taken into account in producing these operating
instructions, the manufacturer accepts no responsibility for any errors or omissions. This
includes any liability for damage that could arise from possible incorrect operation based on this
information. Subject to changes without notice as result of technical development.

SERVICE UND SUPPORT

CONTENTS

TABLE OF CONTENTS

1PREPARATION 3
1.1 UNPACKING THE HUMACYCLER 3
1.2 LOCATING THE HUMACYCLER 4
1.3 ENVIRONMENTAL CONDITIONS 4
1.4 CONNECTING THE INSTRUMENT TO THE COMPUTER 4
1.5 SOFTWARE INSTALLATION 5

2 START THE INSTRUMENT 7

3 SET UP DYES, SAMPLES, PLATES AND PROGRAMS 9

3.1 ABSOLUTE QUANTIFICATION 9
3.2 SINGLE-NUCLEOTIDE POLYMORPHISM (SNP) ANALYSIS 12
3.3 RELATIVE QUANTIFICATION 12

4 RUN PROGRAM 13

5 RESULT ANALYSIS 15

6CAUTION 21
Preparation 3

1 PREPARATION

1.1 Unpacking the HumaCycler

-- Remove the instrument from the shipping container
-- Prove the completeness of the accessories based on the packing list
-- Remove the plastic bag from the instrument
-- Place the instrument in an appropriate location (see 1.2. Locating the
instrument)

Before starting, the Fixing Pin shall be taken out from of the instrument by turn-
ing it anticlockwise (see figure 1).

Figure 1

1 Fixing Pin on the right side

of the HumaCycler

Insert the pin into the unlocker port at its back and fully tighten it clockwise (see
figure 2).

Figure 2

1 Fixing Pin in the unlocker

port

For safety reasons, we recom-

mend that the lifting and un-
1
packing of the instrument should
be performed by two people.
 4

1.2 Locating the HumaCycler

-- Ensure a 30 cm space around the instrument
-- Avoid jam of air inlet of the instrument
-- Position the instrument to allow easy access to the flip button and power
connection
-- Place the instrument in a low dust environment
-- Select a smooth and even surface

1.3 Environmental conditions

-- Maximum altitude: 2000 m
-- Ambient temperature: 10...30C (
-- Maximum humidity: 70%
-- Power supply: 100...240 V; 50&60 Hz, 600 W

1.4 Connecting the instrument to the computer

-- Verify that the power switch button at the back of the instrument is turned
off
-- Attach the power cable to the power entry module
-- Plug the instrument into a properly grounded socket
-- Install the communication box to the interface of the instruments back
-- Connect the communication interface to the computer

Three communication boxes are provided: RS232C, USB and Bluetooth (see
figure 3). If using USB, the USB driver of the provided software must be installed.
If using Bluetooth, Bluetooth equipment and software is needed. The communi-
cation box RS232C can be used directly.

Figure 3
RS232C, Bluetooth and
USB Converter Box.

HumaCycler | Quick Guide

Preparation 5

1.5 Software installation

The provided HCy4 software is specific for each instrument and should not be
used for other instruments.

To install the software, insert the provided CD into the disc drive of the
computer, click onto the HCy4 software file and follow the instructions to install
it.

Figure 4
 6

HumaCycler | Quick Guide

START the instrument 7

2 START THE INSTRUMENT

Press the flip button at the back of the instrument (ON position) and press the
Run Switch button at the instruments front. The button indicator lamps (Error
and Running) blink once. A beep indicates that the instrument is performing a
self test. The mode run switch is merely used for temporary or short tie closing
control system. During the standby mode, the flip button at the back of the in-
strument must be switched off.

To start a run, the instrument has to be connected to the HCy4 software.

Click Instrument Connect Automatically Connect

Usually, it takes a few minutes in which the communication port is searched

automatically by the software. If the communication fails, the error message
Communication Failure will pop up. In this case, verify that the communication
wire is properly fixed and whether another program of the computer occupies
the port (see figure 5).

Figure 5

After the software started, the User Login interface appears. Please enter
User name: admin, Password: admin and click OK (see figure 6).
 8

Figure 6

Click Service User Management Add User and enter your login data

The accessories provide an Anti-Condensate Water Seal Ring for improving the
accuracy of the experiment results and preventing the tubes at and near the
edges of the heating block to generate condensate, which would lead to a re-
duced fluorescent signal. In addition, the seal ring insulates the heat after it is
placed in position. It is applicable for the use of single PCR tubes, 8-strip PCR
tubes, half- skirted and non skirted PCR plates. Full skirted plates cannot be used
with the seal ring. Before starting the experiment, the seal ring should be placed
around the heating block (see figure 7)

Figure 7

HumaCycler | Quick Guide

SET UP DYES, SAMPLES, PLATES AND PROGRAMS 9

3 SET UP DYES, SAMPLES, PLATES AND PROGRAMS

3.1 Absolute Quantification

Click Absolute Quantification, the interface for set up a new absolute quantifi-
cation experiment opens (see figure 8).

Figure 8

1. Set up Detectors
Click Detector
Enter the experiment properties, experiment name (is generated automatically
but can be modified), the user name and add comments to the experiment.

Click Add Detector to add a new target. Enter the name of the target and choose
the reporter dye. If you are using a Human Real-Time PCR Assay (HIV-1, HCV,
HBV, CMV), choose FAM.

Click Add Detector to add another target. Enter the name of the target and
choose the reporter dye. If you are using a Human Real-Time PCR Assay (HIV-1,
HCV, HBV, CMV), choose HEX for the Internal Control (IC) (see figure 9).

Figure 9
 10

2. Set up the sample information

Click Sample, enter the sample ID and press Enter to add the sample to the sam-
ple ID list. If you want to perform batch testing Batch Add can be chosen. Enter
the start sample ID and the number of samples, click Add and samples will be
added automatically.

3. Set up the plate

Click Plate

Select a well on the plate for your sample

Select the corresponding sample on the left side

Select the assay item and set up the properties of your sample (Unknown, Stand-
ard, Non-template Control and Positive Control) and the Concentration Units.
For Human Real-Time PCR assays select IU/ml (see figure 10).

Figure 10

HumaCycler | Quick Guide

SET UP DYES, SAMPLES, PLATES AND PROGRAMS 11

4. Set up the program

Click Program and a default temperature profile will pop up (see figure 11).

Figure 11

To modify the temperature profile, the following modifications can be used:

Add Stage
Hold Stage
PCR Stage
Melting Stage
Infinite Stage
Add Step (before or after an existing step)
Cycles
Liquid Quantity of the Test
Temperature
Time
Measuring Step
Delete
 12

An example of a modified cycling profile is shown (see figure 12).

Figure 12

The set up can be saved as template file.

Click Save As Save as Template

The file will be stored in the template folder.

3.2 Single-Nucleotide Polymorphism (SNP) Analysis

The set up steps for the SNP analysis are basically the same as those for the
Absolute Quantification. There is no Add or Cancel assay setup in the test items.
The program can only add test items and select filter of allele 1 and allele 2.

3.3 Relative Quantification

The set up steps for the Relative Quantification program are basically the same
as those for the Absolute Quantification. There is no Add or Cancel assay set-
up in the test items. For experiments with monochrome tubes, check the sam-
ple information on the left side for the same sample during analysis and select
standard comparison samples. During the analysis, select an analysis method in
options of analysis settings.

HumaCycler | Quick Guide

Run Program 13

4 RUN PROGRAM
To ensure getting better application curves, it is necessary to adjust the gain
of the instrument before the amplification reaction. View the gain in the Gain
Settings under Tools. Select Custom Gain to set the gain by yourself. The default
gain is 7.
To adapt users reagents, the Custom Gain is adopted. Prepare the sample set up
like it is described in the User Manual. Edit the program, sample settings and the
temperature profile as follows:
Temperature 55C
Time 10 sec
Cycles 10

Click Run and Start Run. The gain setting interface appears (see figure 13).
Choose Custom Gain and click OK. Observe the fluorescence curves of the sam-
ples.
Figure 13

The fluorescence values should be between 1000 and 2000. If the values are too
high, repeat the run and decrease the gain volume for the corresponding dye. If
the values are lower than expected, repeat the run and increase the gain volume
for the corresponding dye. If the fluorescence values are in the expected range,
set up the program, samples and plate for the testing.
Fluorescence, temperature curves and the program can be observed during the
run by choosing the corresponding item. Furthermore, the run status is shown,
e.g. start and end time, current cycle and step (see figure 14).

Figure 14
 14

HumaCycler | Quick Guide

Result Analysis 15

5 RESULT ANALYSIS
After the run is completed, an interface to save the experiment will pop up. Click
Yes to save or No to view the experiment but not to save it.

The fluorescence curves and the plate are shown automatically (see
figure 15). To modify the analysis settings, click Analysis Settings. After modifi-
cation click Apply Analysis Settings to save it.

Figure 15

Figure 16
 16

Click Result Summary to view all values for the corresponding wells.

1. Absolute Quantification analsysis

All concentrations of unknown samples are calculated automatically with a
standard curve. The standard curve of the assay is generated based upon the
CT values and concentrations of the used standards. To show the results of the
standard curve, click Standard Curve.

Figure 17

2. Melting Curve analysis

For the Melting Curve analysis click Derivative Curve or Fluorescence Curve to
see the different illustrations of the melting curve (see figure 18).
Figure 18

HumaCycler | Quick Guide

Result Analysis 17

Figure 19

3. Relative Quantification analysis:

The sample of the test tube should be identified as the same bar. Click Relative
Quantification to show the histogram or Amplification Plot to show the fluores-
cence curves (see figure 20).

Figure 20
 18

4. SNP analysis:
Click SNP to show the Allele Scatter Diagram or click Amplification Plot to show
the fluorescence curves (see figure 21).

Figure 21

HumaCycler | Quick Guide

Result Analysis 19
 20

HumaCycler | Quick Guide

6 CAUTION
1. If the instrument will not be used for a long time, the power plug shall be
withdrawn and the instrument shall be covered with dustproof cloth.
2. The instrument surface shall not be cleaned with corrosive cleaning agents
the instrument shall be cleaned with soft cloth soaked with mild cleaner.
3. According to the frequency of using and the working environment of the
instrument, the module shall be regularly cleaned.
4. The PCV socket from the accessories is to paste the information of the
HUMAN authorized local Technical Service. The place to paste it is chosen
by the user.
5. The rubber typhoon from the accessories is to blow dust out of the wells
regularly. Do not press strongly when cleaning the wells in order to avoid
that the optic components of the wells are damaged. In addition, avoid scrat-
ching the surface of the optic components of the well.
HUMAN
Gesellschaft fr Biochemica und Diagnostica mbH
Max-Planck-Ring 21 65205 Wiesbaden Germany
Tel.: +49 6122/9988 0 Fax: +49 6122/9988 100
eMail: human@human.de www.human.de