Haynes Lab:Notebook/David Barclay Undergrad Training/Plasmid Transformation/Entry Base: Difference between revisions
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Protocol: <br> | Protocol: <br> | ||
Using Qiaga Protocol,shown below. <br> | Using Qiaga Protocol,shown below. <br> | ||
[[Image:Lab01.jpg|200px|]] | [[Image:Lab01.jpg|200px|]] <br> | ||
Miniprep followed exactly <br> | Miniprep followed exactly <br> | ||
=====Pre-Miniprep===== | =====A. Pre-Miniprep===== | ||
1. Add 3 mL LB into 15 mL tube <br> | 1. Add 3 mL LB into 15 mL tube <br> | ||
2. Add one colony of KAH06 into 3mL LB Broth: Incubate 7 hrs at 37 C. | 2. Add one colony of KAH06 into 3mL LB Broth: Incubate 7 hrs at 37 C. | ||
=====Miniprep===== | =====B. Miniprep===== | ||
1. Pipette 1.5 mL of sample into 2 mL tube. Spin for 3 min @ max setting (13.1g). Repeat once more to spin entire sample. <br> | 1. Pipette 1.5 mL of sample into 2 mL tube. Spin for 3 min @ max setting (13.1g). Repeat once more to spin entire sample. <br> | ||
NOTE: REMEMBER TO MARK TUBES <br> | NOTE: REMEMBER TO MARK TUBES <br> | ||
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8. Add 400 μL of Zyppy wash buffer into column. Centrifuge 30 secs @ 13.1g. <br> | 8. Add 400 μL of Zyppy wash buffer into column. Centrifuge 30 secs @ 13.1g. <br> | ||
9. Transfer column into new 1.5 mL tube. Add 30 μL of Zyppy Buffer and let sit for 1 min. Centrifuge 15 secs @ 13.1 g. <br> | 9. Transfer column into new 1.5 mL tube. Add 30 μL of Zyppy Buffer and let sit for 1 min. Centrifuge 15 secs @ 13.1 g. <br> | ||
=====DNA Concentration- Take 3 Plate===== | =====C. DNA Concentration- Take 3 Plate===== | ||
1. Open program Gen 5 2.0 before machine. <br> | 1. Open program Gen 5 2.0 before machine. <br> | ||
2. Add 2 μL of sample into holes. <br> | 2. Add 2 μL of sample into holes. <br> | ||
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4. Open program "Nucleic Acid Quantification" <br> | 4. Open program "Nucleic Acid Quantification" <br> | ||
5. Use DI water and kim wipe to clean plate. <br> | 5. Use DI water and kim wipe to clean plate. <br> | ||
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====Results==== | |||
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1.17 μg/μL. Need between 20-80 μg/μL. <br> | |||
0.5 260/280. Need about 1.8 260/280. Shows major contamination. <br> | |||
====Causes of Errors==== | |||
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*Not exactly 600 μL of initial LB+sample. +-50 μL. '''Step A.2.''' <br> | |||
*Pipette did not grab exactly 2 μL to add to Take 3 Plate. '''Step B.2''' <br> | |||
*Buffer used was Zyppy, not Water. Take 3 Plate could not have compensated. ''' Step B.3''' <br> | |||
*High contamination from disturbed pellet? '''Step B.5''' <br> | |||
*Other Contamination in Parts A, B and C. <br> | |||
Revision as of 12:50, 30 June 2013
Project name | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page |
Tuesday, June 25thSteps for TransformationUsing KAH06 for Plasmid.
Friday, June 28thMiniprepAims: Run plasmids through miniprep in order to isolate plasmid DNA and determine concentration. Protocol: A. Pre-Miniprep1. Add 3 mL LB into 15 mL tube B. Miniprep1. Pipette 1.5 mL of sample into 2 mL tube. Spin for 3 min @ max setting (13.1g). Repeat once more to spin entire sample. C. DNA Concentration- Take 3 Plate1. Open program Gen 5 2.0 before machine. Results1.17 μg/μL. Need between 20-80 μg/μL. Causes of Errors
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