Presentation is loading. Please wait.

Presentation is loading. Please wait.

Introduction to CLC Main Workbench 20 June, 2012 Ansuman Chattopadhyay, PhD Head, Molecular Biology Information Services Health Sciences Library System.

Similar presentations


Presentation on theme: "Introduction to CLC Main Workbench 20 June, 2012 Ansuman Chattopadhyay, PhD Head, Molecular Biology Information Services Health Sciences Library System."— Presentation transcript:

1 Introduction to CLC Main Workbench 20 June, 2012 Ansuman Chattopadhyay, PhD Head, Molecular Biology Information Services Health Sciences Library System University of Pittsburgh

2 Sequence Analysis Software Suits Wisconsin GCG VectorNTI DNA STAR-LaserGene Geneious CLC Main

3 Why CLC Main ? Windows Mac Linux DNA, RNA, Protein, Microarray Data Analysis Regular Update HSLS Licensed

4 CLC Main Access HSLS CLC Main Registration  Link: Access via Pitt - Network Connect  Instruction video:

5 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

6 CLC Main Graphical User Interface (GUI)

7 CLC Main

8 Basic Navigation -DNA -Protein

9 Import a DNA Sequence

10 DNA Sequence Human PLCg1  Refseq no: NM_  FASTA file  Raw sequence CLC features: Search, Import, Create new sequence

11

12 CLC DNA sequence

13 Import a Protein Sequence

14 Protein Sequence Human PLCg1  Refseq no: NP_  Uniprot Accession Number: P19174  FASTA file  Raw sequence CLC features: Search, Import, Create new sequence

15 CLC protein sequence

16 Protein sequence manipulation Create a new protein with PLCg1 SH2-SH2- SH3 domains

17 Back Translation Reverse Translate PLCg1 SH2-SH2-SH3

18 Perform Restriction Digestion

19 Restriction Mapping

20 Restriction Digestion

21 Protein Primary Structure Analysis

22 Antigenicity Plot

23 Protein Analysis Report

24 Protease Digestion

25 Proteolytic Cleavage

26 Primer Design

27 Primer Analysis & Design A little something to get you in the mood…

28 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 Kary Mullis

29 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

30 PCR – non specific bands Christiane B etal.,

31 Design PCR Primers to amplify the region covering exons 4-5 in human PLCg1 mRNA sequence

32

33

34 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

35 In silico cloning

36 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

37

38 In silico cloning

39 Sequence Alignment Pair-wise Alignment  Global  Local Multiple Sequence Alignment

40 Sequence Alignment

41 Pair-wise Sequence Alignment

42 Multiple Sequence Alignment

43 PLCg1 Orthologous sequences PLCg1:  Mouse: NP_  Rat: NP_  Cow: NP_  Dog: XP_  Zebra fish: NP_  Human: NP_  NP_067255,NP_037319,NP_776850,XP_542998,NP_919388,NP_

44 Thank you! Any questions? Carrie IwemaAnsuman Chattopadhyay


Download ppt "Introduction to CLC Main Workbench 20 June, 2012 Ansuman Chattopadhyay, PhD Head, Molecular Biology Information Services Health Sciences Library System."

Similar presentations


Ads by Google