From 2007.igem.org

[edit] Growing colonies in broth:

• 1. Prepare 5mL LB + Amp broth in a sterile Falcon tube.
• 2. Pick up a colony from the plate by a micropipette tip or sterile toothpick and put it in the falcon.
• 3. Incubate at 37C incubator for 14-16 hours.

Miniprep Plasmid Isolation (with Qiagen kit):

• 1. Centrifuge the falcons at 4000rpm for 4-10 minutes.
• 2. Resuspend pelleted bacterial cells in 250uL Buffer P1(kept in +4C) and transfer to a 1.5mL eppendorf.
• 3. Add 250uL(microliter) Buffer P2 and invert the tube for ~6 times to mix (do not vortex). Solution should become blue (if indicator is added).
• 4. Add 350uL Buffer N3 and invert the tube immediately for ~6 times. Solution should become wtite and cloudy.
• 5. Centrifuge for 10 minutes at 13000 rpm. A compact white pellet will be formed.
• 6. Pour the supernatant to Qiagen spin column and centrifuge the column for 1 min. Discard the flow-through.
• 7. Wash the spin column by adding 0.75mL Buffer PE and centrifuge for 1 min.
• 8. Discard the flow-through and centrifuge for an additional 6.5-7min to remove residual ethanol in the wash buffer.
• 9. Place the Qiagen prep column in a clean 1.5 mL eppendorf. Add 32uL (can be modified according to the concentration aimed to be obtained) Buffer EB or water to the C center of each Qiagen prep spin column and let stand for 5-10 minutes. Centrifuge for 1 min.

Store the minipreps at -20. The concentration obtained can be measured by Nanodrop.

---

[edit]

Digestion:

For Vector: Mix (to a total of 20 uL): 7-7.5 uL mini-prepped vector DNA 7.5 uL distilled water 0.3 uL restriction enzyme 1 at 20 units/uL 0.3 uL restriction enzyme 2 at 20 units/uL 0.4 uL calf intestinal alkaline phosphatase (CIP) to prevent re-ligation of the vector to itself 2 uL 10x BSA 2 uL 10x appropriate NEB buffer (check from www.neb.com Double Digest Finder)

Insert: Mix (to a total of 10 uL): 7 uL mini-prepped 'vector' DNA 8.2 uL distilled water 0.4 uL restriction enzyme 1 at 20 units/uL 0.4 uL restriction enzyme 2 at 20 units/uL 2 uL 10x BSA 2 uL 10x appropriate NEB buffer

• Incubate overnight at 37C.
• Gel purify the insert using a Qiagen kit. Elute using 20 uL.
• PCR purify the vector (can be gel purified too). Elute in 20 uL.

Ligation (with Roche Rapid Ligation kit): Mix: 1 uL digested vector 3 uL digested insert

• The optimum molar ratio is 1:3, the volumes can be modified according to concentrations of the vector and insert. For sticky end ligations 1:5 ratio can be used.
• Complete to 10 uL with 1X reagent 2 of the kit (diluted from 5X with distilled water), Vortex and spin.
• Add 10 uL reagent #1.
• Add 1 uL reagent #3.
• Ligate for 10 minutes at room temperature.
• Transform 2 uL of ligation mix in 25 uL DH5alpha competent cells.

Transformation

• Thaw competent E. coli in ice. Take 25uL cells in prechilled eppendorfs. Slowly add 2uL plasmid DNA.
• Incubate in ice for 30 minutes (or more).
• Heat shock at 42C water bath for 30 seconds (timing is critical).
• Incubate for 5 min in ice