1. Lecture 7
January 16, 2016
Biotech 3

2. Lecture Topics 6. PCR Review Blue-White Colony Screen
Mathematical concept and cycle number
Polymerase rate and fidelity
Primer design for point mutations
PCR Parameters: DNA sequence, primers, type of polymerase, Tm, extension time, number of cycles
Transformations
Competent cells
4. Standard Operating Procedure Review
5. Primer Design Part II
Start codon
In frame
Stop codon
Designing tags

3. β-galactosidase β-galactosidase Oxidation Dimerization β-galactosidase
5-Bromo-4-Chloro-3-indoyl-β-D-galactopyranoside
(X-gal)
5-Bromo-6-Chloro-3-indoxyl
5,5'-dibromo-4,4'-dichloro-indigo
Absorbs at 615 nm
Blue Precipitate
β-galactosidase
β-D-galactose
o-Nitrophenol
Orthonitrophenalgalctopyranoside (ONPG)

4. LacZ Gene Disruption – Blue White Colony Screening

5. βlue-White Colony Screen
Today’s experiment: Transformations
Sunday
Saturday 1/23

6. Edvotek 300 Flowchart

7. Transformations - 1
Transformation introducing exogenous genetic material into a bacterial cell.
Methods:
- Calcium chloride

8. Transformations - 2
Transformation introducing exogenous genetic material into a bacterial cell.
Methods:
- Electroporation

9. Calculating Transformation Efficiency
DNA Spread on the plate (μg) =
For example:
50 ng of plasmid DNA Transformed into 500 μl of cells 10 μl of the cells are spread onto a plate 60 CFUs are observed
10 μl X 0.05 μg
DNA Spread on the plate (μg) =
500 μl
Transformation Efficiency
60 CFU =
0.001 μg =
60 X 104 CFU/ μg

10. NdeI and XhoI Restriction Sites

11. Cell Competency

12. Bacterial Transformation

13. Bacterial Transformation – Heat Shock

14. Electroporation

15. Standard Operating Procedure
1. Title Page
Title of the procedure
SOP identification number
Date of issue or revision
Name of the agency/division/branch to where the SOP applies
Signatures of those who prepared and approved of the SOP
Table of Contents
Quality Assurance/Quality Control
Performance evaluation samples
Reference
Cite all significant references.
Scope and applicability
Purpose of the process, limits, standards, etc.
Methodology and procedures
List all steps with necessary details including what equipment is needed.
Cover sequential procedures
Terminology
Identify acronyms and abbreviations that are not commonly known
Health and Safety
Equipment and supplies
Complete list of what is required to perform the procedure
10. Cautions and interferences
Cover what could go wrong and what may interfere with the final product
11. Each page needs to have control document notation (Title, ID#, revision number, date, page #)