User:Derek J. Sanchez/Notebook/TB Sensor - MS applied project/2012/03/07

From OpenWetWare
Jump to navigationJump to search
TB Sensor Main project page
Previous entry      Next entry

03/07/12


10:00AM, Creating Liquid Culture

The cultured plates have finished and the results are as follows:

  • Plate 1 had one colony
  • Plate 2 had no growth
  • Plate 3 had three colonies
  • Plate 4 had many colonies (possibly over grown)
  • Plate 5 had no growth (Amp control)
  • Plate 6 had no growth (Kan control)

The three plates with successful colonies were then used for liquid cultures.

The liquid cultures were labeled as follows:

Tube Label Plate Label BioBrick part number Origin Well Plasmid Backbone Organism Resistance Description
1 1 BBa_J06504 From Yue pSB1A2 Bacteria Amp mCherry (mRFP1), works better then original registry part
2 3 BBa_K274100 2011 Plate 3 6H pSB1A2 Used in E. coli Amp E. chromi (Red)
3 4 BBa_J06702 From Yue pSB1T3 E. coli Amp E. chromi (Violet)


The liquid cultures were then prepared.

  1. Three culture tubes were labeled 1-3 and set into a rack.
  2. Using a sterile pipette 2mL of LB broth was added to each tube.
    The LB broth contained 100μg/mL of Amp
  3. A sterile pipette tip was used to touch the center of the colony on plate 1 and dropped into tube 1
  4. A sterile pipette tip was used to touch the center of a colony on plate 3 and dropped into tube 2
  5. A sterile pipette tip was used to touch the center of a colony on plate 4 and dropped into tube 3
  6. The tubes were then capped, loosely, and placed in the warm room (37°C).
    In the tubes were put at an angle (for small samples) on to a rotating rack.
  7. The plates were then sealed around the edge with paraffin tape and stored in the cold room (4°C).

The cultures should be left in these conditions for approximately 8 hours.

5:00PM

Extracting DNA plasmid using QIAprep. The liquid cultures that were created this morning will have the DNA plasmid removed. this procedure was done following the directions out of the protocol book, some of the directions have been altered to fit the needs of the lab.

Equipment:

  • 2mL tube one per culture.
  • P1 buffer, it contains RNase A
  • P2???
  • N3
  • QIAprep Tube
  • PE Buffer


Directions:

  1. The liquid cultures were transferred into 2 mL tubes that were labeled 1-3, via pipette.
  2. The tubes were then placed in the centrifuge and ran for 3 minutes at 17.9 g
  3. Remove form centrifuge and pour off liquid
    The pellet is sticky and stays on the bottom of the tube.
  4. 250μL of P1 was added to each tube.
  5. Tubes were then vortexed until pellet was again suspended in liquid.
  6. 250μL of P2??? was added the tubes were mixed by inversion gently 6 times.
    The sample will then turn blue as a pH indicator.
  7. 350μL of N3 was added and the solution was inverted 10 times.
    Invert until no blue color remains. a white substance forms (excess cell parts).
  8. The solution is then centrifuged for 10 minutes at 17.9 g.
  9. Label QIAprep Tube, label the blue inner part.
  10. The liquid was then poured into the labeled QIAprep tube.
    The DNA in the fluid will be collected in the filter.
  11. The tubes were then centrifuged for 1 minute at 17.9 g.
  12. The flow through was then discarded.
  13. 75μL of PE Buffer was then pipetted over the filter
  14. The tubes were then centrifuged for 1 minute at 17.9 g.
  15. The tube was then centrifuged again and the flow through was discarded.
  16. Steps 13-15 were then repeated a second time.

More instructions to follow.

A agar plate was also created from the pipette tips in the original tubes. The plate was labeled with: Resistance, Strain, Initials, and Date. Then each of the three samples on the one plate were labeled.





Retrieved from "https://openwetware.org/mediawiki/index.php?title=User:Derek_J._Sanchez/Notebook/TB_Sensor_-_MS_applied_project/2012/03/07&oldid=1009861"