Team:Wash U/Protocol

text

Transformation

1. Start thawing the competent cells on crushed ice.

2. Add 50 µL of thawed competent cells and then 1 - 2 µL of the resuspended DNA to the labelled tubes. Make sure to keep the competent cells on ice.

3. Incubate the cells on ice for 30 minutes.

4. Heat shock the cells by immersion in a pre-heated water bath at 42ºC for 60 seconds. A water bath improves heat transfer to the cells.

5. Incubate the cells on ice for 5 minutes.

6. Add 200 μl of SOC broth (make sure that the broth does not contain antibiotics and is not contaminated)

7. 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 plasmids with antibiotic resistance other than ampicillin.

8. Label two petri dishes with LB agar and the appropriate antibiotic(s) with the part number, plasmid, and antibiotic resistance. Plate 20 µl and 200 µl of the transformation onto the dishes, and spread. This helps ensure that you will be able to pick out a single colony.

9. Incubate the plate at 37ºC for 12-14 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 inactivate the antibiotic outside of the bacteria.

Note: Restriction sites E=EcoR1-HF; X=Xba1; S=Spe1; P=Pst1; M=Mixed Site

To view the full BioBrick Manual procedures, please click here.

Biobrick Assembly Overview

1. Begin BioBrick Assembly with three separate plasmids, an upstream part, a down stream part, and a destination plasmid. It is important that the destination plasmid contain the toxic gene ccdB in the BioBrick cloning site and a different antibiotic resistance to the upstream and downstream parts.

2. Digest each part with the proper restriction enzymes to isolate the upstream and downstream parts, and to remove the BioBrick from the destination plasmid.

3. Ligate the products. The result will be the upstream part connected to the downstream part in the destination plasmid. This new composite part can then be used to transform competent cells.

To view the full Biobrick Assembly manual, please click here.

Digestion

text

Ligation

text

Gel Electrophoresis

text

Recipes

Lysogeny Broth (LB) Media

• 10g Tryptone
• 5g Yeast Extract
• 10g NaCl
• 15g Agar
• up to 1 Liter H20

ATCC medium: Van Niel's yeast agar

• 1.0 g K2HPO4
• 0.5 g MgSO4
• 10.0 g yeast extract
• 20.0 g agar
• 1.0 L tap water
• Adjust pH to 7.0-7.2

Super Optimal Broth (SOB)

• 2% w/v bacto-tryptone (20g)
• 0.5% w/v bacto-yeast extract (5g)
• 8.56mM NaCl (0.5g) or 10mM NaCl (0.584g)
• 2.5mM KCl (0.186g)
• ddH2O to 1L

Super Optimal Broth with Catabolite repression (SOC)

• In addition to all of the SOB components,
• 10mM MgCl2 (0.952g)
• 20mM glucose (3.603g)

50X TAE Buffer

• 242 g Tris base (2-amino-2-hydroxymethyl-propane-1,3-diol) (= 2 mole)
• 57.1 ml glacial acetic acid (= 100% acetic acid) (57.19 ml = 1 mole)
• 100 ml 0.5 M Na2 EDTA (pH 8.0)
• ddH2O to 1L

5X TBE Buffer

• 53 g of Tris base (CAS# 37186)
• 27.5 g of boric acid (CAS# 11280)
• 20 ml of 0.5 M EDTA (CAS# 60004) (pH 8.0)
• ddH2O to 1L

Tris EDTA (TE) buffer 5X 1L

• 750 mL d-H20
• 242 g Tris base
• 57.1 mL glacial acetic acid
• 100 mL 0.5M EDTA (93.05 g EDTA in 500 mL d-H20, pH ~8.0)
• fill to 1 L
• adjust pH to 8.5

Agarose Gel (for electrophoresis of DNA >100bp)

• 50 mL 1X TAE buffer
• 0.8 g agarose
• 2.5 microliters EtBr (Caution: EtBr is a known carcinogen)
• Note: Jacob suggested adding 1.0 microliter EtBr to gel and 3.0 microliters to TAE buffer in rig.