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| |style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span> | | |style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Haynes BioBrick Cloning</span> |
| |style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html> </html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}} | | |style="background-color: #F2F2F2" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]] }}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}} |
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| <!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> | | <!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> |
| ==Tuesday, June 25th==
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| ==== Steps for Transformation ====
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| ----
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| Using KAH06 for Plasmid.<br>
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| Take 1 μL of KAH06.<br>
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| Add 9 μL of ddH20. <br>
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| ----
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| ==Friday, June 28th==
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| ====Miniprep====
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| ----
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| Aims: Run plasmids through miniprep in order to isolate plasmid DNA and determine concentration.
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| Protocol: <br>
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| Using Qiaga Protocol,shown below. <br>
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| [[Image:Lab01.jpg|200px|]] <br>
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| Miniprep followed exactly <br>
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| =====A. Pre-Miniprep=====
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| 1. Add 3 mL LB into 15 mL tube <br>
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| 2. Add one colony of KAH06 into 3mL LB Broth: Incubate 7 hrs at 37 C.
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| =====B. Miniprep=====
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| 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>
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| NOTE: REMEMBER TO MARK TUBES <br>
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| 2. Take 600 μL of sample. Add 100 μL 7x lysis buffer (blue) and invert 4 times. Time limit 2 mins for lysis. <br>
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| 3. Add 350 μL Neutralization Buffer (yellow) invert until sample become yellow (10 times). Neutralization Buffer must be refrigerated. <br>
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| 4. Centrifuge 2 mins @ 13.1g. <br>
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| 5. Transfer ~850 μL into spin column. Put spin column into collection tube. Centrifuge 15 secs @ 13.1g. Discard flow through. <br>
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| 7. Add 200 μL of elution buffer (ENDO Wash Buffer). Centrifuge 15 secs @ 13.1g. <br>
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| 8. Add 400 μL of Zyppy wash buffer into column. Centrifuge 30 secs @ 13.1g. <br>
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| 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>
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| =====C. DNA Concentration- Take 3 Plate=====
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| 1. Open program Gen 5 2.0 before machine. <br>
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| 2. Add 2 μL of sample into holes. <br>
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| 3. Use either water or buffer as blank depending on buffer DNA was added to. <br>
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| 4. Open program "Nucleic Acid Quantification" <br>
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| 5. Use DI water and kim wipe to clean plate. <br>
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| ----
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| ====Results====
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| ----
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| 1.17 μg/μL. Need between 20-80 μg/μL. <br>
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| 0.5 260/280. Need about 1.8 260/280. Shows major contamination. <br>
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| ----
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| ====Causes of Errors====
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| *Not exactly 600 μL of initial LB+sample. +-50 μL. '''Step A.2.''' <br>
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| *Pipette did not grab exactly 2 μL to add to Take 3 Plate. '''Step B.2''' <br>
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| *Buffer used was Zyppy, not Water. Take 3 Plate could not have compensated. ''' Step B.3''' <br>
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| *High contamination from disturbed pellet? '''Step B.5''' <br>
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| *Other Contamination in Parts A, B and C. <br>
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