2×2×96well Microplate Box Type Rotor 

Overview

• DNA can be isolated and detected from as little as 100 µL of sputum
• Effective removal of PCR inhibitors
• Compatible with Norgen’s Saliva Collection and Preservation Device (Cat. RU49000)
• Sputum Liquification Buffer (Cat. 28289) can be purchased separately

This kit constitutes an all-in-one system for the rapid isolation of DNA from sputum samples. It allows for the isolation of bacterial or eukaryotic DNA from the sputum samples using spin-column chromatography based on Norgen’s proprietary resin. The kit includes all the reagents needed to process sputum DNA. The protocol can be completed in 30 minutes. Purified DNA is of the highest quality and free from inhibitors, and can be used in sensitive downstream applications including PCR and qPCR.

Background

A sputum specimen is the name given to the mucus which is expectorated from the lower airways. High quality sputum samples should contain very little saliva, as this may contaminate the sputum sample with oral bacteria. Sputum samples are typically evaluated to look for infections such as Moraxella catarrhalis, Mycobacterium tuberculosis, Streptococcus pneumoniae ;and Haemophilus influenza. Other pathogens can be detected in sputum as well, including HIV. In addition, sputum samples are useful in the detection of lung cancer or to evaluate chronic inflammation.

Details

Supporting Data

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Kit Specifications
Time to Complete DNA Isolation	< 1 hour
Average Yield of DNA from 1.0 mL*	75 μg
Average Purity (OD260/280)	1.8 – 2.1

* Average DNA yield will vary depending on the health status of the donor

Storage Conditions and Product Stability
All solutions should be kept tightly sealed and stored at room temperature. This kit is stable for 2 years after the date of shipment.

Norgen’s Sputum DNA Isolation Kit contains ready-to-use Proteinase K, which is dissolved in a specially prepared storage buffer. The buffered Proteinase K is stable for up to 2 years after the date of shipment when stored at room temperature.

Component	Cat. 46200 (25 preps)
Slurry D	55 mL
Proteinase K in Storage Buffer	0.6 mL
Solution WN	4 mL
Wash Solution BE	9 mL
Elution Buffer B	8 mL
Mini Filter Spin Columns	25
Collection Tubes	25
Elution Tubes (1.7 mL)	25
Product Insert	1

endo-BCN-PEG8-amine is a PEG linker featuring a BCN and a primary amine. The BCN group is a click chemistry handle which quickly reacts with azides while the primary amine can be used to form amides with carboxylic acids in the presence of coupling reagents such as HATU or EDC.

endo-BCN-PEG8-amine is a PEG linker featuring a BCN and a primary amine. The BCN group is a click chemistry handle which quickly reacts with azides while the primary amine can be used to form amides with carboxylic acids in the presence of coupling reagents such as HATU or EDC.

Overview

• Protocol optimized for DNA isolated from a diversity of samples including stool, soil, water, saliva, plant, urine, skin, and more
• Simple and quick workflow: library could be prepared in less than 5 hours
• Component of Norgen’s metagenomics workflow
• A single NGS run can be prepared with up to 384 unique dual-index libraries

Sample type purification kit guide 

The 16S V3-V4 Library Preparation Kit for Illumina consists of the reagents and components required for library preparation of the 16S V3-V4 amplicon libraries to be used for next-generation sequencing on Illumina platforms. All molecular reagents including primers, enzyme mixes, indexes, and buffers are provided. Instructions for PCR clean up with the AMPure XP Magnetic Beads (supplied by customer) are also included for rapid purification of nucleic acid products generated at two steps of the workflow. The library prep workflow could be used for purified DNA inputs from different sources including stool, soil, water, saliva, plant, urine, skin swab, vaginal swab, cheek swab, nasal swab, plasma/serum, tongue swab, gum swab, and others.

The 16S V3-V4 Library Preparation Kit for Illumina has a streamlined procedure that reduces the handling time such that the library prep procedure can be completed in approximately 4 hours (see diagram below). Input DNA is first subjected to targeted PCR to amplify the V3-V4 region of the DNA encoding 16S rRNA. The post-PCR reaction is then cleaned up using AMPure XP beads. Dual index primers are then added using a limited-cycle PCR. The indexed amplicons flanked by 5′ and 3′ barcoded adaptors are then cleaned using AMPure XP beads. The libraries are then ready for quantification, pooling and sequencing.

Details

Minimum amount of starting material:	2.5 µL of DNA (5 ng/µL)
Time to complete library preparation:	4 hours

Storage Conditions and Product Stability
Norgen’s 16S V3-V4 Library Prep Kit for Illumina is shipped as one kit box (for the 24 prep kit) or two sub-component kits (for the 96 prep kit). All kits should be stored at -20°C upon arrival.

All kit components should remain stable for at least 1 year when stored at the specified storage conditions.

Step	Component	Cat. 70400 (24 preps)	Cat. 70410, 70420, 70430, 70440 (96 preps)
Amplicon PCR (PCR 1)	MGX Master Mix	330 µL	1,320 µL
	16S V3-V4 Primer Mix	70 µL	280 µL
Index PCR (PCR 2)	Indexing Master Mix	660 µL	2 x 1,320 µL
	N7xx Index Primer	50 µL	50 µL
	S5xx Index Primer	70 µL	70 µL
PCR Clean-Up	Resuspension Buffer	2 x 1,250 µL	2 x 5,000 µL
	Nuclease-free water	1,250 µL	1 x 6,000 µL