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

Revision as of 19:22, 11 September 2015

Project
- Design
- Parts
- Code
- Judging
Team
Outreach
Notebook

Protocols

Recipes

LB-Agar Plates

LB Broth

SOC Media

3A Assembly

Transformation

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

Materials

2µl resuspended DNA (Resuspend well in 10ul dH20, pipette up and down several times, let sit for a few minutes)

Competent cells (50ul per transformation)

Ice (in ice bucket/container)

2ml tube (1 per a transformation')

42ºC water bath

SOC media

1 small LB plate and 1 large LB+antibiotic per transformation

Spreader

37ºC incubator
Procedure

Start thawing the competent cells on ice.
Add 50 µL of thawed competent cells into pre-chilled 2ml tube, and another 50µL into a 2ml tube, labelled for your control.
Add 2 µL of the resuspended DNA to the 2ml tube. Pipet up and down a few times, gently. Make sure to keep the competent cells on ice.
Close tubes and incubate the cells on ice for 30 minutes.
Heat shock the cells by immersion in a pre-heated water bath at 42ºC for 60 seconds.
Incubate the cells on ice for 5 minutes.
Add 200 μl of SOC media (make sure that the broth does not contain antibiotics and is not contaminated) to each transformation
Incubate the cells at 37ºC for 2 hours while the tubes are rotating or shaking. Important: 2 hour recovery time helps in transformation efficiency, especially for plasmid backbones with antibiotic resistance other than ampicillin.
Label two petri dishes with LB agar and the appropriate antibiotic(s) with the part number, plasmid backbone, and antibiotic resistance. 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.
Spread remaining transformation onto labeled LB agar plate and spread, approx 50µl.
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.
You can pick a single colony, make a glycerol stock, grow up a cell culture and miniprep.

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

Revision as of 19:22, 11 September 2015

Project

Team

Outreach

Notebook

Protocols

Recipes

LB-Agar Plates

LB Broth

SOC Media

3A Assembly

Transformation

Miniprep

Restriction Digest

Ligation

Gel Electrophoresis

Gel Extraction

PCR

PCR Cleanup

Making Color

Luciferase

Chromoproteins