Course outline. 4. 4. Plant Growth Promoting Rhizobacteria- Our research focus. 5. 5. Estimation of bacterial population in soil. 8. 6. Isolation of bacterial strains.
Bacterial Identification and Metagenomics 2013
COURSE MANUAL M. SAJJAD MIRZA, SOHAIL HAMEED, GHULAM RASUL, FATHIA MUBEEN, ASMA IMRAN, AHMAD ZAHEER, MUTHER MANSOOR QAISRANI Culturable Bacteria
Non-culturable Bacteria Metageno mics
Novel orga nism
Novel Gene s
New Antib
New Enzy mes
? And More
Pure Culture DNA
Soil DNA
Soil
PCR16S rDNA
PCR 16S rDNA
Meta –omics Metagenomics, Metaproteomics,
Numerous Bacteria Detected
Few Bacteria Identified
Phylogenetic Analysis
Sequenc ing
Sequencing
Phylogenetic Analysis
National Institute for Biotechnology and Genetic Engineering (NIBGE) P. O. Box 577 Jhang Road, Faisalabad, PAKISTAN Tel: +92 41 2651475-79, Fax: +92 41 2651472 Web: www.nibge.org
© 2013 National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan.
All rights reserved. No part of this publication should be reproduced, stored in a retrieval system or transmitted in any form or by any means electronic, mechanical, photocopying, recording or otherwise, without the prior written permission of the publisher, National Institute for Biotechnology and Genetic Engineering (NIBGE), P. O. Box 577, Faisalabad, Pakistan. Written by: M. Sajjad Mirza, Sohail Hameed, Ghulam Rasul, Fathia Mubeen, Asma Imran, Ahmad Zaheer, Muther Mansoor Qaisrani
P.O. Box 577 Jhang Road, Faisalabad PAKISTAN Tel: (92)+041-2651475-79 Fax: (92)+041-2651472
No responsibility is assumed by the National Institute for Biotechnology and Genetic Engineering (NIBGE) for any injury and /or damage to the persons or property as a matter of products liability, negligence or otherwise, or from by use or operation of any methods, products, instructions or ideas contained in the material herein.
ISBN: 978-969-8189-20-4
BACTERIAL IDENTIFICATION AND METAGENOMICS March 4-8, 2013
TRAINING COURSE MANUAL
M. Sajjad Mirza, Sohail Hameed, Ghulam Rasul, Fathia Mubeen, Asma Imran, Ahmad Zaheer, Muther Mansoor Qaisrani
National Institute for Biotechnology and Genetic Engineering (NIBGE) P. O. Box 577 Jhang Road, Faisalabad, PAKISTAN Tel: +92 41 2651475-79, Fax: +92 41 2651472 Web: www.nibge.org PAKISTAN ATOMIC ENERGY COMMISION (PAEC)
Table of Contents Sr. No.
Contents
Page No.
1
NIBGE-an over view
1
2
Course introduction
3
3
Course outline
4
4
Plant Growth Promoting Rhizobacteria- Our research focus
5
5
Estimation of bacterial population in soil
8
6
Isolation of bacterial strains
10
7
MPN (Most Probable Number) of nitrogen fixing bacteria in soil
11
8
Isolation of nitrogen fixing bacteria
13
9
Isolation of DNA from bacterial cultures
16
10
Phenol-chloroform precipitation (for further purification of DNA)
17
11
Agarose gel electrophoresis
18
12
Protocol for extraction of DNA from soil
19
13
Polymerase chain reaction (PCR)
21
14
Sequence analysis of 16S rRNA for identification of bacteria
22
15
rep-PCR Fingerprinting
25
16
Extraction of DNA from agarose gel
27
17
Ligation of PCR Fragment into pTZ57R/T vector
28
18
Preparation of competent cells (E. coli)
29
19
Transformation by Heat shock method
30
20
Isolation of E. coli plasmid DNA by alkaline lysis
31
21
Data Analysis and Interpretation
33
22
Reactions, chemicals and Media used
34
a) LB (Luria-Bertini) Medium
34
b) Antibiotics stock
34
c) Digestion reactions for confirmation
34
d) Digestion reactions for differentiation in strain
34
e) TA cloning Vector pTZ57R/T
35
f) 1 kb DNA Ladder
35
23
References
36
ISO-9001-2008 Certified Organisation
National Institute for Biotechnology and Genetic Engineering (NIBGE) P. O. Box 577 Jhang Road, Faisalabad, PAKISTAN Tel: +92 41 2651475-79, Fax: +92 41 2651472 Web: www.nibge.org PAKISTAN ATOMIC ENERGY COMMISION (PAEC)
