Team:Johns Hopkins/Notebook

Experiments

Protocols

Plasmid Extraction

Reagents:

1. Buffer P1 - Resuspension Buffer
   50mM Tris-Cl, pH 8.0, 10mM EDTA, 100ug/mL RNase A
   Storage condition - 4oC after adding RNase A
   Prep - Dissolve 6.06g Tris base, 3.72g EDTA-2H20 in 800mL dH20. Adjust the pH to 8.0 with HCl.
   Adjust the volume to 1 liter with dH2O. Add 100mg RNase A per liter of P1.
2. Buffer P2 - Lysis Buffer
   200mM NaOH, 1% SDS
   Storage condition - RT
   Dissolve 8.09g of NaOH pellets in 950mL dH2O, 50mL 20% SDS solution.
   The final volume should be 1 liter.
3. Buffer N3 - Neutralization Buffer for spin columns.
   Composition unknown
   Storage condition - RT
4. PB Buffer - Binding Buffer
   Composition Unknown (Proprietary)
   Storage condition - RT
5. Buffer PE - Wash Buffer
   Composition unknown
   Storage condition - RT

Protocol:

1. Innoculate 3-4 mL of the E. coli cells in glass tubes with the right selection resistance in LB (Luria Bertani) medium.
2. Transfer solution to a 2 mL Eppendorf tube and spin down at max speed (~13,000 rpm) for 30 seconds.
3. Pour out LB medium and repeat until all of the solution from the innoculation tube is pelleted at the bottom of the Eppendorf tube. Each spin down after the first requires one full minute.
4. Re-suspend the pelleted E. coli in 250 µL Buffer P1 and transfer solution to micro-centrifuge tube provided by the Qiaprep Kit. (This can be the same tube as the 2 mL Eppendorf tube) This step requires a strong vortex in order to fully re-suspend the pelleted cells. May require 3-4 minutes of continuous agitation.
5. Add 250 µL of Buffer P2 and mix gently by inverting the tube 4-6 times. Be very gentle! Shear stresses can ruin the experiment after the lysis of cells from P2 Buffer.
6. Add 350 µL of Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times.
7. Spin down at max speed for 10 minutes. A white pellet near the bottom and side of the walls will form.
8. Extract the supernatant and add 500 µL of PB buffer. Place mixture into the Qiaprep spin column.
9. Centrifuge mixture for 1 minute, max speed.
10. Remove liquid at bottom.
11. Add 750 µL of PE Buffer to Qiaprep spin column.
12. Centrifuge mixture for 1 minute, max speed.
13. Remove liquid at bottom and centrifuge again for 1 minute at max speed to remove more of the PE buffer.
14. Transfer Qiaprep spin column to sterile Eppendorf tube and add 50 µL of dH2O and let the spin column equilibrate for 10 minutes with the dH2O.
15. Spin down column for 1 minute, max speed.
16. Confirm size with gel electrophoresis with 1% agarose gel and appropriate DNA Ladder. (Remember circular plasmids can become supercoiled and produce smaller apparent sizes on the gel)
17. Use Nanodrop™ to confirm DNA concentration.

Yeast Transformation

To transform a desired plasmid into a host yeast cell.
Date: 8/25/10 Used to transform ref-GFP

Reagents:

1. PEG-TE LiOAc
   10 mL 10x TE
   10 mL 1 M LiOAc
   80 mL 50% PEG (3350 mw)
2. TE-LiOAc

Results

Notebook

You should make use of the calendar feature on the wiki and start a lab notebook. This may be looked at by the judges to see how your work progressed throughout the summer. It is a very useful organizational tool as well.