User:Perry/Summer 2006 Harvard iGEM/Protocols

Gel electrophoresis
For x% gel, mix x grams Ultrapure agarose in 100ml 1X TBE in a plastic flask, heat by microwave and swirl with top screwed on loosely until clear, cool, added with 3ul (with gloves), pour into gel frame, let set for 20 min. Place into gel running box and submerge barely with 1X TBE. Load wells, and run gel at appropriate voltage and time.

Loading dye is BTB dye 50% glycerol + 10X TBE. Dye has been added to 1X 1kb ladder at 1:11 ratio; 10ul used.

Gel extraction
Qiagen gel extraction pages 25-26

Ligation
Roche Rapid DNA ligation kit page 2

Transformation
Chemically competent OneShot Top10 cells. Let cells thaw on ice. Add appropriate amount of DNA, tap gently to mix, and let sit on ice for 20 minutes. Heat shock for 30 sec at 42dC; let cool on ice for 2 min. Add 250ul SOC media, and shake for 1 hour at 37dC. Pipet onto agar plate treated with appropriate drug, and spread. Leave in 37dC overnight with agar side up.

Miniprep
Qiagen protocol

Digest
x ul DNA

10% by volume buffer (10X, choose buffer best for enzymes used)

1% BSA (100X)

2% enzyme 1

2% enzyme 2

y ul water (to fill to desired mixture volume)

Mix and incubate for 1-2hr (or overnight if you want) at 37dC. Then, if you plan on storing, incubate for 20min at 80dC. This kills the restriction enzymes.