Difference between revisions of "Team:NYU Shanghai/Protocols"

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Revision as of 05:27, 14 September 2015

• 

• Project

  • Design
  • Parts
  • Code
  • Judging

• Team

  • Members
  • Partners
  • Attributions

• Outreach

  • Collaborations
  • Practices
  • Virtual Reality

• Notebook

  • Daily Notes
  • Protocols
  • Bibliography

Protocols

Recipes

LB-Agar Plates

This is how you make an agar plate.

LB Broth

This is how you make LB Broth.

SOC Media

This is how you make SOC media.

3A Assembly

Transformation

Estimated time: 3 hours (plus 14-18 hour incubation)

Materials

2µl resuspended DNA

Competent cells (50ul per transformation)

Ice

2ml tube

42ºC water bath

SOC or LB media

1 LB plate and 1 LB+antibiotic per transformation

Spreader

37ºC incubator

Shaker

Procedure

1. Thaw competent cells on ice.
2. Add 50µL of thawed competent cells into pre-chilled 2ml tube.
3. Add 2µL of resuspended DNA to the 2ml tube. Pipet up and down a few times, gently. Keep the competent cells on ice.
4. Close tubes and incubate the cells on ice for 30 minutes.
5. Heat shock the cells by immersion in a pre-heated water bath at 42ºC for 60 seconds.
6. Incubate the cells on ice for 5 minutes.
7. Add 200 μl of SOC or LB media to each transformation
8. Incubate the cells at 37ºC for 2 hours at 260rpm in the shaker.
9. Label petri dishes with the appropriate antibiotic with the part number, plasmid backbone, antibiotic resistance, and date. Plate 200 µl of the transformation onto the labeled antibiotic dishes, and spread. This helps ensure that you will be able to pick out a single colony.
10. Spread remaining transformation onto labeled LB agar plate and spread, approx 50µl.
11. Incubate the plates at 37ºC for 12-18 hours, making sure the agar side of the plate is up. If incubated for too long the antibiotics start to break down and un-transformed cells will begin to grow. This is especially true for ampicillin - because the resistance enzyme is excreted by the bacteria, and inactivates the antibiotic outside of the bacteria.
12. You can pick a single colony, make a glycerol stock, grow up a cell culture and miniprep.

Controls

Competent cells on LB plate

Competent cells on LB + antibiotic plate

Competent cells + plasmid on LB plate

Miniprep

This is how you miniprep.

Restriction Digest

This is how you do restriction digest.

Ligation

This is how you do ligation.

Gel Electrophoresis

This is how you do gel electrophoresis.

Gel Extraction

This is how you do gel extraction.

PCR

This is the conditions we used for PCR.

PCR Cleanup

This is the conditions we used for PCR cleanup.

Making Color

Luciferase

This is the conditions we used to express luciferase.

Chromoproteins

This is the conditions we used to express chromoproteins.

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