Haynes Lab:Notebook/David Barclay Undergrad Training/Plasmid Transformation/Entry Base

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Tuesday, June 25th

Steps for Transformation


Using KAH06 for Plasmid.
Take 1 μL of KAH06.
Add 9 μL of ddH20.



Friday, June 28th

Miniprep


Aims: Run plasmids through miniprep in order to isolate plasmid DNA and determine concentration.

Protocol:
Using Qiaga Protocol,shown below.

Miniprep followed exactly

A. Pre-Miniprep

1. Add 3 mL LB into 15 mL tube
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.
NOTE: REMEMBER TO MARK TUBES
2. Take 600 μL of sample. Add 100 μL 7x lysis buffer (blue) and invert 4 times. Time limit 2 mins for lysis.
3. Add 350 μL Neutralization Buffer (yellow) invert until sample become yellow (10 times). Neutralization Buffer must be refrigerated.
4. Centrifuge 2 mins @ 13.1g.
5. Transfer ~850 μL into spin column. Put spin column into collection tube. Centrifuge 15 secs @ 13.1g. Discard flow through.
7. Add 200 μL of elution buffer (ENDO Wash Buffer). Centrifuge 15 secs @ 13.1g.
8. Add 400 μL of Zyppy wash buffer into column. Centrifuge 30 secs @ 13.1g.
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.

C. DNA Concentration- Take 3 Plate

1. Open program Gen 5 2.0 before machine.
2. Add 2 μL of sample into holes.
3. Use either water or buffer as blank depending on buffer DNA was added to.
4. Open program "Nucleic Acid Quantification"
5. Use DI water and kim wipe to clean plate.


Results


  • 1.17 μg/μL. Need between 20-80 μg/μL.
  • 0.5 260/280. Need about 1.8 260/280. Shows major contamination.

Causes of Errors


  • Not exactly 600 μL of initial LB+sample. +-50 μL. Step A.2.
  • Pipette did not grab exactly 2 μL to add to Take 3 Plate. Step B.2
  • Buffer used was Zyppy, not Water. Take 3 Plate could not have compensated. Step B.3
  • High contamination from disturbed pellet? Step B.5
  • Other Contamination in Parts A, B and C.




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