HIT – Heparin-Induced Thrombocytopenia Test (20)

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 V1-V3 Library Preparation Kit for Illumina consists of the reagents and components required for library preparation of the 16S V1-V3 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 V1-V3 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 V1-V3 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

Supporting Data

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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 V1-V3 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. 70200 (24 preps)	Cat. 70210, 70220, 70230, 70240 (96 preps)
Amplicon PCR (PCR 1)	MGX Master Mix	330 µL	1,320 µL
	16S V1-V3 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

Introduction

Intended Use

For the isolation and differentiation of sorbitol-negative Escherichia coli serotype O157 from food and animal feed and other materials

Principle and Interpretation

Enzymatic digest of casein and enzymatic digest of animal tissue provides nitrogen source, vitamins and growth factors; sodium chloride maintains balanced osmoticpressure; sorbitol is a fermentable sugar; No. 3 bile salt and crystal violet inhibit Gram-positive bacteria, potassiumtellurite inhibits non-o157 Escherichia coli, and cefixime inhibits Proteus; neutral red is a pH indicator; and agar is the coagulant of the culture medium.

Formulation

Ingredients	/liter
Enzymatic digest of casein	17.0g
Enzymatic digest of animal tissue	3.0g
Sorbitol	10.0g
Bile salt No.3	1.5g
Sodium chloride	5.0g
Neutral red	0.03g
Crystal violet	0.001g
Agar	15.0g
pH 7.1±0.2 at 25°C

Preparation

Weigh 51.5g of this product, add 1L of distilled water or deionized water, stir, heat and boil until completely dissolved, dispense into Erlenmeyer flasks, and sterilize at 121℃ for 15min. Melt the sterilized culture medium and cool it to about 45℃, add 1 tube of supporting reagent (SR0240) (0.25mg potassium tellurite and 0.005mg Cefixime) to every 100mL of basal culture medium, mix and pour into the plate.

Quality Control

Cultural characteristics observed after incubation at 35-37°C for 18-24hours

Quality control strains	Growth	Colony color
Escherichia coli ATCC25922	PR≥0.7	pale pink to red
Escherichia coli 0157:H7 NCTC12900	PR≥0.7	colorless
Staphylococcus aureus ATCC 25923	Total inhibition	–

Sorage and Shelf Life

Keep container tightly closed, store in a cool, dry place, away from bright light. Storage period of 3 years.

Precautions

1. When weighing the dehydrated medium, please wear masks to avoid causing respiratory system discomfort

2. Keep container tightly closed after using to prevent clumping.

Waste Disposal

Microbiological contamination was disposed by autoclaving at 121°C for 30 minutes.

Intended Use For the isolation and differentiation of sorbitol-negative Escherichia coli serotype O157 from food and animal feed and other materials Principle and Interpretation Enzymatic ……