Difference between revisions of "Haynes Lab:Notebook/David Barclay Undergrad Training/Plasmid Transformation/Entry Base"

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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>
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==Tuesday, June 25th==
==== Steps for Transformation ====
Using KAH06 for Plasmid.<br>
Take 1 μL of KAH06.<br>
Add 9 μL of ddH20. <br>
==Friday, June 28th==
Aims: Run plasmids through miniprep in order to isolate plasmid DNA and determine concentration.
Protocol: <br>
Using Qiaga Protocol,shown below. <br>
[[Image:Lab01.jpg|200px|]] <br>
Miniprep followed exactly <br>
=====A. Pre-Miniprep=====
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.
=====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>
2. Take 600 μL of sample. Add 100 μL 7x lysis buffer (blue) and invert 4 times. Time limit 2 mins for lysis. <br>
3. Add 350 μL Neutralization Buffer (yellow) invert until sample become yellow (10 times). Neutralization Buffer must be refrigerated. <br>
4. Centrifuge 2 mins @ 13.1g. <br>
5. Transfer ~850 μL into spin column. Put spin column into collection tube. Centrifuge 15 secs @ 13.1g. Discard flow through. <br>
7. Add 200 μL of elution buffer (ENDO Wash Buffer). Centrifuge 15 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>
=====C. DNA Concentration- Take 3 Plate=====
1. Open program Gen 5 2.0 before machine. <br>
2. Add 2 μL of sample into holes. <br>
3. Use either water or buffer as blank depending on buffer DNA was added to. <br>
4. Open program "Nucleic Acid Quantification" <br>
5. Use DI water and kim wipe to clean plate. <br>
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====
*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>

Latest revision as of 22:53, 26 September 2017

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