Himedia Insta NX User manual

The information contained herein is believed to be accurate and complete. However no warranty or guarantee whatsoever is made
or is to be implied with respect to such information or with respect to any product, method or apparatus referred to herein
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15
U n z i p p i n g G e n e s
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MBIN008 Insta NX® Fungal DNA Purification Kit
Kit Contents
Product Code Reagents provided 12 Preps
DS0401 Fungal DNA Extraction Cartridges 12 nos.
DS0337 1 ml tip set 12 nos.
DS0016 Lysis Buffer (PL) 6 ml
DS0017 Precipitation Buffer (PS) 1.8 ml
DS0003 RNase A Solution (20 mg/ml) 300 µl
DS0040 Elution Buffer (ET) [10 mM Tris-Cl, pH 8.5] 350 µl
DBCA04 HiBead Tubes 12 nos.
DSCA02 HiShredder (in DBCA016 Collection Tube) 12 nos.
PW1139 Collection Tubes, Polypropylene (2.0 ml) 40 nos.
DBCA021 Columns for Insta NX 12 nos.
Introduction
This kit provides a fast and easy method to purify total DNA from fungal samples for
downstream applications such as PCR, Southern blotting technique etc. The DNA purification
procedure using the column comprises of three steps viz. adsorption of DNA to the Super- S
membrane, removal of residual contaminants and elution of pure genomic DNA. The Super- S
columns have a high binding capacity and high quality genomic DNA is obtained from various
species.
Insta NX® Fungal DNA Purification Kit
This kit simplifies isolation of DNA from various fungal samples with vacuum based innovative
Super- S column technology. The Super- S columns contains specially developed membranes for
optimal binding of genomic DNA. After the initial binding of DNA, impurities like proteins,
polysaccharides, low molecular weight metabolites and salts are removed by short washing
steps. High quality DNA is finally eluted in the Elution Buffer provided with the kit.
The salient features of this kit are as follows:
a. Cross- Contamination free
b. 12 samples can be processed at a time.
c. Prefilled reagent cartridge
d. High purity, high yield, as good as manual.
Elution
The yield of genomic DNA depends on the sample type and the number of cells in the sample.
An elution with 100 µl of Elution Buffer (ET) will provide sufficient DNA to carry out multiple
amplification reactions. Elution with volume less than 100 µl will increase the final DNA
concentration, but will reduce the overall DNA yield. The eluted DNA ranges in size upto 20-30
kb, and is suitable for direct use in PCR, restriction digestion and Southern blotting applications.

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Concentration, yield and purity of DNA
Spectrophotometric analysis and agarose gel electrophoresis will reveal the concentration and
the purity of the genomic DNA. Use Elution Buffer (ET) to dilute samples and to calibrate the
spectrophotometer, measure the absorbance at 260 nm, 280 nm, and 320 nm using a quartz
micro cuvette.
Absorbance readings at 260 nm should fall between 0.1 and 1.0. The 320 nm absorbance is used
to correct for background absorbance. An absorbance of 1.0 at 260 nm corresponds to
approximately 50 µg/ml of DNA. The A260 -A320 /A280 -A320 ratio should be 1.6-1.9. Purity is
determined by calculating the ratio of absorbance at 260 nm to absorbance at 280 nm. DNA
purified by Insta NX® Fungal DNA Purification Kit is free of protein and other contaminants that
can inhibit PCR or other enzymatic reactions.
Concentration of DNA sample (µg/ml) = 50 x A260 x dilution factor.
Storage
Store the Insta NX® Fungal DNA Purification Kit between 15-25°C except certain components as
specified on each labels. Under recommended condition kit is stable for 4 months.
Materials needed but not provided
Tabletop Microcentrifuge (with rotor for 2.0 ml tubes) capable of upto ≈14,000 rpm
(LA1001)
65°C water bath or heating block
Vortex Mixer
Insta NX® (LA1056)
Molecular Biology Grade Water (ML024)
Micropipettes (LA617, LA613) and Tips (LA974)
General Preparation Instructions
IMPORTANT: Please go through the instruction manual before starting the experiment.
1. Preheat a water bath or heating block to 65°C.
RNase A enzyme treatment
RNase A is a type of RNase that is commonly used in research. RNase A (e.g. bovine pancreatic
ribonuclease A) is one of the sturdiest enzymes in common laboratory usage. It cleaves 3' end
of unpaired C and U residues.
Unit Definition for RNase A
One unit of the enzyme causes an increase in absorbance of 1.0 at 260 nm when yeast RNA is
hydrolyzed at 37°C and pH 5.0. Fifty units are approximately equivalent to 1 Kunitz unit. It is
completely free of DNases and proteases. The specific activity is 90 U/mg.
The product as supplied is stable at room temperature (15-25°C).

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Procedure (For Non- Barcode Mode)
1. Turn on Insta NX® instrument. Allow the instrument to initialize.
2. Once the instrument is ready, select “Login” on the instrument screen.
Fig. 1
3. Enter the Password to login.
Fig. 2
4. On the Home screen, select “ Start Purification”
Fig. 3

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5. On the Product Type screen, select the type of purification to be carried out.
Fig. 4
6. On the Setting screen
a. Select Elution volume: 50/ 100/150 or 200µl
b. Select Kit Name: Select the type of extraction procedure to be performed.
Fig. 5
7. Lift open the front door and take out the 12-in-1 rack for preparation.
NOTE: You can follow Step by Step Setup worktable for preparation of 12-in-1 rack
8. Place the Fungal DNA Extraction Cartridges (DS0401) on 12-in-1 rack as shown in the
following figure.
Fig. 6
MBIN008 Insta NX® Fun
g
al DNA

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9. Place the columns (DBCA021) on the cartridge as shown below:
Fig. 7
10. Place 1ml tip (DS0337) set onto the 12-in-1 rack.
Fig. 8
11. Open the metal lid and place the Collection tube, Polypropylene (PW1139) into 12-in-1 rack
(as shown in figure below) and close the metal lid.
Fig. 9

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12. Place the 12-in-1 rack into the instrument as shown in the below fig. and secure the rack in
position by two lock plates aside the worktable.
Fig. 10
13. Prepare sample with proper Pre-treatment
Sample Pre-treatment procedure
Weigh 150 mg of the fungal material and add to the HiBead tubes (DBCA04).
Add 400 l of Lysis Solution (PL) (DS0016).Vortex vigorously for 5 minutes.
Add 20 l of RNaseA Solution and incubate the mixture for 10 minutes at 65°C.
Centrifuge the samples at 14,000 rpm for 1 minute at room temperature (15-25°C).
Transfer the supernatant to a new collection tube and add 130 l of Precipitation Buffer
(PS) (DS0017). Mix and incubate in ice for 5 minutes.
Transfer the entire lysate on to the HiShredder (DSCA02) and centrifuge the samples at
14,000 rpm for 1 minute at room temperature (15-25°C).
Transfer the flow through without disturbing the pellet to a new Collection tube (provided)
Proceed with step 14 for DNA purification in Insta NX® Machine as follows.
14. Place the Collection tube containing sample onto the 12-in-1 rack.
Fig. 11

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15. Close the front door. Click Start Run.
Fig. 12
16. Following screen will appear while the run is going on.
Fig. 13
17. After the run is completed, the screen will look like the below figure:
Fig. 14
18. Lift the instrument lid and take out the 12-in-1 rack. Remove the elution tubes and discard
all other plastic wares.
Run complete
MBIN008 Insta NX® Fun
g
al DNA Purification

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19. The eluted DNA can be further used for PCR amplification.
Representative image of PCR set up in HiMedia’s PCR Machines
Storage of the eluate with purified DNA: The eluate contains pure DNA. For short-term storage
(24-48 hrs.) of the DNA, 2-8°C is recommended. For long-term storage, -20°C or lower
temperature (-80°C) is recommended. Avoid repeated freezing and thawing of the sample
which may cause denaturing of DNA. The Elution Buffer will help to stabilize the DNA at these
temperatures.
Precautions
Read the procedure carefully before starting the experiment.
Performance and Evaluation
Each lot of Insta NX® Fungal DNA Purification Kit is tested against predetermined specifications
to ensure consistent product quality.
Quality Control
Type of Sample DNA Yield DNA Purity
Fungal sample 4-12 g 1.6-1.9
Troubleshooting Guide
Sr. No. Problem Possible Cause Solution
1. Poor / Lower yield
of genomic DNA
Sample is old Yield of genomic DNA varies from sample
to sample. It is necessary to carry out
enrichment procedure properly as per
the protocol.
Insufficient lysis The amount of the starting material can
be reduced or the amount of buffers PL
and PS can be increased
Fig. a: Insta Q 96-PLUS Fig. b: Wee Machine

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Safety Information
Take appropriate laboratory safety measures and wear gloves when handling. Not compatible
with disinfecting agents containing bleach. Please refer the Safety Data Sheet (SDS) for
information regarding hazards and safe handling practices.
Disposal
User must ensure safe disposal by autoclaving and/or incineration of used or unusable
preparations of this product. Follow established laboratory procedures in disposing of
infectious materials and material that comes into contact with clinical sample must be
decontaminated and disposed off in accordance with current laboratory techniques.
Technical Assistance
At HiMedia, we pride ourselves on the quality and availability of our technical support. For any
kind of technical assistance, mail to [email protected].
PIMBIN008_O/0619 MBIN008-04