Table Of Contents
BIT 295 Lab Schedule
Table of Contents
List of Protocols in Protocols.io
Biotechnology & Sustainability Research Protocols
Protocol Acknowledgements and References
Lab Safety and Personal Protective Equipment
Pipetting 1
Overview and Goals 1
Preparation 2
Procedure 3
Activity 1 4
Activity 2 5
Critical Thinking Questions for Pipetting 6
Plate Streaking 7
Overview and Goals 7
Preparation 8
Procedure 9
Clean Up Instructions 10
Critical Thinking Questions for Plate Streaking 11
Preparing Biolog Growth Plates 12
Overview and Goals 12
Use the Protocols.io copy of the procedure accessed here to run and check off each step of the procedure. 12
Preparation 13
Procedure 14
Activity 1: Multichannel pipetting 14
Activity 2: Turbidimeter 15
Activity 3: Biolog plate preparation 16
Clean Up Instructions 17
Critical Thinking Questions for Preparing Biolog Growth Plates 18
Staining and Microscopy – Protocol 1: LIVE BacLight 19
Overview and Goals 19
Preparation 20
Note Before You Begin: 20
Procedure 21
Bacterial Staining 21
Clean-Up Instructions 22
Critical Thinking Questions Microscopy 23
Staining and Microscopy – Protocol 2: MycoLight 24
Overview and Goals 24
Preparation 25
Note Before You Begin: 25
Procedure 26
Preparation of Bacterial Samples (done by instructors) 26
Staining Protocol 26
Clean-Up Instructions 27
Critical Thinking Questions Microscopy 28
Bacterial Isolate Genomic DNA Isolation – ONT NO-MISS 29
Overview and Goals 29
Preparation 30
Procedure 30
Use the Protocols.io copy of the procedure accessed here to run and check off each step of the procedure. 30
Note Before You Begin: 30
Safety 30
Supplies 30
Materials 30
Consumables 30
Equipment 30
Bacterial Lysis and Genomic DNA Isolation 31
Growth of Isolates 31
Bacterial Lysis 31
gDNA Binding and Elution 31
Critical Thinking Questions for Bacterial Genomic DNA Isolation 32
Bacterial Genomic DNA Isolation – QIAGEN PowerSoil Pro 33
Overview and Goals 33
Preparation 34
Procedure 34
Use the Protocols.io copy of the procedure accessed here to run and check off each step of the procedure. 34
Note Before You Begin: 34
Bacterial Lysis and Genomic DNA Isolation 35
Growth of Isolates 35
Bacterial Lysis 35
gDNA Binding and Elution 35
Critical Thinking Questions for Bacterial Genomic DNA Isolation 36
Bacterial Genomic DNA Isolation – NEB HMW 37
Overview and Goals 37
Preparation 38
Procedure 38
Bacterial Lysis and Genomic DNA Isolation 38
Bacterial Lysis: 38
HMW gDNA Binding and Elution: 38
Note Before You Begin: 39
Activity 1: Bacterial Lysis 40
Activity 2: HMW gDNA Binding and Elution 41
Critical Thinking Questions for Bacterial Genomic DNA Isolation 42
Quantifying and Checking Genomic DNA 43
Overview and Goals 43
Preparation 44
Use the Protocols.io copy of the procedure accessed here to run and check off each step of the procedure. 44
Note Before You Begin: 44
Required Materials: 44
Activity 1: Quantification of DNA with the Implen Spectrophotometer 45
Recording you DNA quantifications 45
Activity 2: DNA Quantification with Qubit 46
Activity 3: Genomic DNA Assessment with TapeStation 47
Critical Thinking Questions for Quantifying and Checking Genomic DNA 48
16S Gene PCR Amplification and Sanger Sequencing 49
PCR amplification of 16S 49
Preparation 50
Use the Protocols.io copy of the procedure accessed here to run and check off each step. 50
Note Before You Begin: 50
PCR Clean-Up 51
Sanger Sequencing Sample Preparations 52
Analyzing Chromatograms to Confirm Consensus Sequence 53
BLAST 16S sequence to identify bacterial species or related species. (Performed during synchronous session) 53
Rapid Barcode Sequencing with the Nanopore MinION 54
Overview and Goals 54
Equipment and Consumables 55
Oxford Nanopore Technologies (ONT) Kit and Flow Cells: 55
All consumables: 55
All equipment: 55
Materials: 55
Consumables: 55
Preparation 56
Protocol 56
Nanopore Rapid Barcoding protocol on Protocols.io 56
Library Preparation 57
Instrument Setup and Flow Cell Priming (in parallel with sample prep) 58
Critical Thinking Questions for Nanopore Sequencing 59
References
Laboratory Supplies
Pipetting & Plate Streaking
Preparing Biolog Growth Plates
