![Page 1: Introduction to CLC Main Workbench 20 June, 2012 Ansuman Chattopadhyay, PhD Head, Molecular Biology Information Services Health Sciences Library System](https://reader036.vdocument.in/reader036/viewer/2022062312/551c5850550346b1458b50e4/html5/thumbnails/1.jpg)
Introduction to CLC Main Workbench20 June, 2012
Ansuman Chattopadhyay, PhDHead, Molecular Biology Information ServicesHealth Sciences Library SystemUniversity of [email protected]
![Page 2: Introduction to CLC Main Workbench 20 June, 2012 Ansuman Chattopadhyay, PhD Head, Molecular Biology Information Services Health Sciences Library System](https://reader036.vdocument.in/reader036/viewer/2022062312/551c5850550346b1458b50e4/html5/thumbnails/2.jpg)
Sequence Analysis Software Suits
Wisconsin GCG VectorNTI DNA STAR-LaserGene Geneious
CLC Main
![Page 3: Introduction to CLC Main Workbench 20 June, 2012 Ansuman Chattopadhyay, PhD Head, Molecular Biology Information Services Health Sciences Library System](https://reader036.vdocument.in/reader036/viewer/2022062312/551c5850550346b1458b50e4/html5/thumbnails/3.jpg)
Why CLC Main ?
Windows Mac Linux DNA, RNA, Protein, Microarray Data Analysis Regular Update HSLS Licensed
![Page 4: Introduction to CLC Main Workbench 20 June, 2012 Ansuman Chattopadhyay, PhD Head, Molecular Biology Information Services Health Sciences Library System](https://reader036.vdocument.in/reader036/viewer/2022062312/551c5850550346b1458b50e4/html5/thumbnails/4.jpg)
CLC Main Access
HSLS CLC Main Registration Link: http://www.hsls.pitt.edu/molbio/clcmain
Access via Pitt - Network Connect Instruction video: http://goo.gl/JNjMt
![Page 5: Introduction to CLC Main Workbench 20 June, 2012 Ansuman Chattopadhyay, PhD Head, Molecular Biology Information Services Health Sciences Library System](https://reader036.vdocument.in/reader036/viewer/2022062312/551c5850550346b1458b50e4/html5/thumbnails/5.jpg)
Topics
CLC Main GUI Import DNA sequence into CLC Import Protein sequence into CLC Design PCR primers Perform restriction enzymes digestions Run in silico agarose gels Protein primary structure analysis Protease digestions
![Page 6: Introduction to CLC Main Workbench 20 June, 2012 Ansuman Chattopadhyay, PhD Head, Molecular Biology Information Services Health Sciences Library System](https://reader036.vdocument.in/reader036/viewer/2022062312/551c5850550346b1458b50e4/html5/thumbnails/6.jpg)
CLC Main Graphical User Interface (GUI)
![Page 7: Introduction to CLC Main Workbench 20 June, 2012 Ansuman Chattopadhyay, PhD Head, Molecular Biology Information Services Health Sciences Library System](https://reader036.vdocument.in/reader036/viewer/2022062312/551c5850550346b1458b50e4/html5/thumbnails/7.jpg)
CLC Main
![Page 8: Introduction to CLC Main Workbench 20 June, 2012 Ansuman Chattopadhyay, PhD Head, Molecular Biology Information Services Health Sciences Library System](https://reader036.vdocument.in/reader036/viewer/2022062312/551c5850550346b1458b50e4/html5/thumbnails/8.jpg)
Basic Navigation
-DNA
-Protein
![Page 9: Introduction to CLC Main Workbench 20 June, 2012 Ansuman Chattopadhyay, PhD Head, Molecular Biology Information Services Health Sciences Library System](https://reader036.vdocument.in/reader036/viewer/2022062312/551c5850550346b1458b50e4/html5/thumbnails/9.jpg)
Import a DNA Sequence
![Page 10: Introduction to CLC Main Workbench 20 June, 2012 Ansuman Chattopadhyay, PhD Head, Molecular Biology Information Services Health Sciences Library System](https://reader036.vdocument.in/reader036/viewer/2022062312/551c5850550346b1458b50e4/html5/thumbnails/10.jpg)
DNA Sequence
Human PLCg1 Refseq no: NM_002660 FASTA file Raw sequence
CLC features:
Search, Import, Create new sequence
![Page 11: Introduction to CLC Main Workbench 20 June, 2012 Ansuman Chattopadhyay, PhD Head, Molecular Biology Information Services Health Sciences Library System](https://reader036.vdocument.in/reader036/viewer/2022062312/551c5850550346b1458b50e4/html5/thumbnails/11.jpg)
http://www.hsls.pitt.edu/molbio
Import nucleotide and protein sequences into CLC Main workbench
Link to the video tutorial:http://media.hsls.pitt.edu/media/clres/.swf
Resources
•CLC Main workbench: http://www.hsls.pitt.edu/molbio/clcmain
![Page 12: Introduction to CLC Main Workbench 20 June, 2012 Ansuman Chattopadhyay, PhD Head, Molecular Biology Information Services Health Sciences Library System](https://reader036.vdocument.in/reader036/viewer/2022062312/551c5850550346b1458b50e4/html5/thumbnails/12.jpg)
CLC DNA sequence
![Page 13: Introduction to CLC Main Workbench 20 June, 2012 Ansuman Chattopadhyay, PhD Head, Molecular Biology Information Services Health Sciences Library System](https://reader036.vdocument.in/reader036/viewer/2022062312/551c5850550346b1458b50e4/html5/thumbnails/13.jpg)
Import a Protein Sequence
![Page 14: Introduction to CLC Main Workbench 20 June, 2012 Ansuman Chattopadhyay, PhD Head, Molecular Biology Information Services Health Sciences Library System](https://reader036.vdocument.in/reader036/viewer/2022062312/551c5850550346b1458b50e4/html5/thumbnails/14.jpg)
Protein Sequence
Human PLCg1 Refseq no: NP_002651 Uniprot Accession Number: P19174 FASTA file Raw sequence
CLC features:
Search, Import, Create new sequence
![Page 15: Introduction to CLC Main Workbench 20 June, 2012 Ansuman Chattopadhyay, PhD Head, Molecular Biology Information Services Health Sciences Library System](https://reader036.vdocument.in/reader036/viewer/2022062312/551c5850550346b1458b50e4/html5/thumbnails/15.jpg)
CLC protein sequence
![Page 16: Introduction to CLC Main Workbench 20 June, 2012 Ansuman Chattopadhyay, PhD Head, Molecular Biology Information Services Health Sciences Library System](https://reader036.vdocument.in/reader036/viewer/2022062312/551c5850550346b1458b50e4/html5/thumbnails/16.jpg)
Protein sequence manipulation Create a new protein with PLCg1 SH2-SH2-
SH3 domains
![Page 17: Introduction to CLC Main Workbench 20 June, 2012 Ansuman Chattopadhyay, PhD Head, Molecular Biology Information Services Health Sciences Library System](https://reader036.vdocument.in/reader036/viewer/2022062312/551c5850550346b1458b50e4/html5/thumbnails/17.jpg)
Back Translation
Reverse Translate PLCg1 SH2-SH2-SH3
![Page 18: Introduction to CLC Main Workbench 20 June, 2012 Ansuman Chattopadhyay, PhD Head, Molecular Biology Information Services Health Sciences Library System](https://reader036.vdocument.in/reader036/viewer/2022062312/551c5850550346b1458b50e4/html5/thumbnails/18.jpg)
Perform Restriction Digestion
![Page 19: Introduction to CLC Main Workbench 20 June, 2012 Ansuman Chattopadhyay, PhD Head, Molecular Biology Information Services Health Sciences Library System](https://reader036.vdocument.in/reader036/viewer/2022062312/551c5850550346b1458b50e4/html5/thumbnails/19.jpg)
Restriction Mapping
http://www.hsls.pitt.edu/molbio
www.biologyreference.com
![Page 20: Introduction to CLC Main Workbench 20 June, 2012 Ansuman Chattopadhyay, PhD Head, Molecular Biology Information Services Health Sciences Library System](https://reader036.vdocument.in/reader036/viewer/2022062312/551c5850550346b1458b50e4/html5/thumbnails/20.jpg)
Restriction Digestion
![Page 21: Introduction to CLC Main Workbench 20 June, 2012 Ansuman Chattopadhyay, PhD Head, Molecular Biology Information Services Health Sciences Library System](https://reader036.vdocument.in/reader036/viewer/2022062312/551c5850550346b1458b50e4/html5/thumbnails/21.jpg)
Protein Primary Structure Analysis
![Page 22: Introduction to CLC Main Workbench 20 June, 2012 Ansuman Chattopadhyay, PhD Head, Molecular Biology Information Services Health Sciences Library System](https://reader036.vdocument.in/reader036/viewer/2022062312/551c5850550346b1458b50e4/html5/thumbnails/22.jpg)
Antigenicity Plot
![Page 23: Introduction to CLC Main Workbench 20 June, 2012 Ansuman Chattopadhyay, PhD Head, Molecular Biology Information Services Health Sciences Library System](https://reader036.vdocument.in/reader036/viewer/2022062312/551c5850550346b1458b50e4/html5/thumbnails/23.jpg)
Protein Analysis Report
![Page 24: Introduction to CLC Main Workbench 20 June, 2012 Ansuman Chattopadhyay, PhD Head, Molecular Biology Information Services Health Sciences Library System](https://reader036.vdocument.in/reader036/viewer/2022062312/551c5850550346b1458b50e4/html5/thumbnails/24.jpg)
Protease Digestion
![Page 25: Introduction to CLC Main Workbench 20 June, 2012 Ansuman Chattopadhyay, PhD Head, Molecular Biology Information Services Health Sciences Library System](https://reader036.vdocument.in/reader036/viewer/2022062312/551c5850550346b1458b50e4/html5/thumbnails/25.jpg)
Proteolytic Cleavage
![Page 26: Introduction to CLC Main Workbench 20 June, 2012 Ansuman Chattopadhyay, PhD Head, Molecular Biology Information Services Health Sciences Library System](https://reader036.vdocument.in/reader036/viewer/2022062312/551c5850550346b1458b50e4/html5/thumbnails/26.jpg)
Primer Design
![Page 27: Introduction to CLC Main Workbench 20 June, 2012 Ansuman Chattopadhyay, PhD Head, Molecular Biology Information Services Health Sciences Library System](https://reader036.vdocument.in/reader036/viewer/2022062312/551c5850550346b1458b50e4/html5/thumbnails/27.jpg)
Primer Analysis & Design
http://www.hsls.pitt.edu/molbio
A little something to get you in the mood…
![Page 28: Introduction to CLC Main Workbench 20 June, 2012 Ansuman Chattopadhyay, PhD Head, Molecular Biology Information Services Health Sciences Library System](https://reader036.vdocument.in/reader036/viewer/2022062312/551c5850550346b1458b50e4/html5/thumbnails/28.jpg)
Polymerase Chain Reaction (PCR) very simple
exponential amplification similar to natural DNA replication
The primary reagents, used in PCR are:
Template DNA–DNA sequence to amplify DNA nucleotides–building blocks for new DNA Taq polymerase–heat stable enzyme catalyzes new DNA Primers–single-stranded DNA, ~20-50 nucleotides,
complimentary to a short region on either side of template DNA
http://www.hsls.pitt.edu/molbio
1983-Kary Mullis
![Page 29: Introduction to CLC Main Workbench 20 June, 2012 Ansuman Chattopadhyay, PhD Head, Molecular Biology Information Services Health Sciences Library System](https://reader036.vdocument.in/reader036/viewer/2022062312/551c5850550346b1458b50e4/html5/thumbnails/29.jpg)
Things to consider for primer design…
Primer-Dimer formation
Secondary Structures in Primers
Illegitimate Priming in Template DNA due to repeated sequences
Incompatibility with PCR conditions
SOURCE: NCBI
http://www.hsls.pitt.edu/molbio
![Page 30: Introduction to CLC Main Workbench 20 June, 2012 Ansuman Chattopadhyay, PhD Head, Molecular Biology Information Services Health Sciences Library System](https://reader036.vdocument.in/reader036/viewer/2022062312/551c5850550346b1458b50e4/html5/thumbnails/30.jpg)
PCR – non specific bands
Christiane B etal., http://goo.gl/KVCxI
http://www.hsls.pitt.edu/molbio
![Page 31: Introduction to CLC Main Workbench 20 June, 2012 Ansuman Chattopadhyay, PhD Head, Molecular Biology Information Services Health Sciences Library System](https://reader036.vdocument.in/reader036/viewer/2022062312/551c5850550346b1458b50e4/html5/thumbnails/31.jpg)
Design PCR Primers to amplify the region covering exons 4-5 in human PLCg1 mRNA sequence
http://www.hsls.pitt.edu/molbio
![Page 32: Introduction to CLC Main Workbench 20 June, 2012 Ansuman Chattopadhyay, PhD Head, Molecular Biology Information Services Health Sciences Library System](https://reader036.vdocument.in/reader036/viewer/2022062312/551c5850550346b1458b50e4/html5/thumbnails/32.jpg)
http://www.hsls.pitt.edu/molbio
Design PCR primers to amplify a region present in a DNA sequence
Link to the video tutorial:http://media.hsls.pitt.edu/media/molbiovideios/PCR.swf
Resources
•CLC Main workbench: http://www.hsls.pitt.edu/molbio/clcmain
![Page 33: Introduction to CLC Main Workbench 20 June, 2012 Ansuman Chattopadhyay, PhD Head, Molecular Biology Information Services Health Sciences Library System](https://reader036.vdocument.in/reader036/viewer/2022062312/551c5850550346b1458b50e4/html5/thumbnails/33.jpg)
http://www.hsls.pitt.edu/molbio
Design primers for TaqMan real-time PCR
Link to the video tutorial:http://media.hsls.pitt.edu/media/molbiovideios/pcr2-clc-ac0112.swf
Resources
•CLC Main workbench: http://www.hsls.pitt.edu/molbio/clcmain
![Page 34: Introduction to CLC Main Workbench 20 June, 2012 Ansuman Chattopadhyay, PhD Head, Molecular Biology Information Services Health Sciences Library System](https://reader036.vdocument.in/reader036/viewer/2022062312/551c5850550346b1458b50e4/html5/thumbnails/34.jpg)
Design PCR primers to amplify a DNA region covering a protein domain
PCR amplification of human PLCg1 SH3 domain
CLC Main Features: Reverse Translate PCR Primer Design
Video Tutorials
http://www.hsls.pitt.edu/molbio
![Page 35: Introduction to CLC Main Workbench 20 June, 2012 Ansuman Chattopadhyay, PhD Head, Molecular Biology Information Services Health Sciences Library System](https://reader036.vdocument.in/reader036/viewer/2022062312/551c5850550346b1458b50e4/html5/thumbnails/35.jpg)
In silico cloning
![Page 36: Introduction to CLC Main Workbench 20 June, 2012 Ansuman Chattopadhyay, PhD Head, Molecular Biology Information Services Health Sciences Library System](https://reader036.vdocument.in/reader036/viewer/2022062312/551c5850550346b1458b50e4/html5/thumbnails/36.jpg)
Molecule Construction
Clone a fragment from pBR322 into pUC19
☼ Donor fragment: pBR322, 5’EcoRI—3’AvaI
☼ Recipient fragment: pUC19, 5’SmaI—3’EcoRI
video tutorials
http://www.hsls.pitt.edu/molbio
![Page 39: Introduction to CLC Main Workbench 20 June, 2012 Ansuman Chattopadhyay, PhD Head, Molecular Biology Information Services Health Sciences Library System](https://reader036.vdocument.in/reader036/viewer/2022062312/551c5850550346b1458b50e4/html5/thumbnails/39.jpg)
Sequence Alignment
Pair-wise Alignment Global Local
Multiple Sequence Alignment
http://www.hsls.pitt.edu/molbio
![Page 43: Introduction to CLC Main Workbench 20 June, 2012 Ansuman Chattopadhyay, PhD Head, Molecular Biology Information Services Health Sciences Library System](https://reader036.vdocument.in/reader036/viewer/2022062312/551c5850550346b1458b50e4/html5/thumbnails/43.jpg)
PLCg1 Orthologous sequences PLCg1:
Mouse: NP_067255 Rat: NP_037319 Cow: NP_776850 Dog: XP_542998 Zebra fish: NP_919388
Human: NP_002651
NP_067255,NP_037319,NP_776850,XP_542998,NP_919388,NP_002651
http://www.hsls.pitt.edu/molbio
![Page 44: Introduction to CLC Main Workbench 20 June, 2012 Ansuman Chattopadhyay, PhD Head, Molecular Biology Information Services Health Sciences Library System](https://reader036.vdocument.in/reader036/viewer/2022062312/551c5850550346b1458b50e4/html5/thumbnails/44.jpg)
Thank you!Any questions?
Carrie Iwema Ansuman [email protected] [email protected] 412-383-6887 412-648-1297
http://www.hsls.pitt.edu/molbio