Team:Chiba/Notebook/protocol

Protocols

• Transformation (Using Zymo comp.)
• DNA_Purification

• Sigma prep
• Zymo DNA Cleam&Concentrator Kit

• Agarose gel electrophoresis
• Digestion
• PCR
• Gel extract
• Dephosphorylation of DNA
• Phosphorylation of DNA
• Ligation
• Time Delay Test

Transformation

Day 1 morning

• 100 ml SOB medium in 1L or 500 mL flask and sterilize

• E.coli culture grown in 2 mL of fresh LB medium.

Day 1 night

• Inoculate preculture (100 μL-1 mL) to sterile SOB medium.Shake culture vigoruoussly at 20-25 ℃ until OD is 0.4-0.6.

Day 2

• Transfer the culture to ice 10min.

• Prepare Wash buffer and Competent buffer by adding 3 mL Dilution buffer to 3 mL of Wash buffer(x2) and to 2.5 mL of Competent buffer(2x), respectively.(on ice)

• Pellet the cells by centrifugarion at 2500rpm for 6 min.

• Remove supernatant and genlly resupend the cells in 6 mL ice-cold Wash buffer(1x).

• Pellet the cells by centrifugarion at 2500rpm for 6 min.

• Completely remove the supernatant and gently resuspend the cells in 6 mL ice-cold Competent buffer(1x).

• Aliquot (on ice) 100μL of cell suspension into sterile 1.5 mL microtube and store in deep freezer.

DNA Purification

Sigma prep

Zymo DNA Cleam&Concentrator Kit

• Add 2 volume of DNA binding buffer to each volume of DNA sample, Use voltex to mix.

• Load mixture silica column and place column into a 2 ml collection tube

• Centrifuge at full speed for 30 sec. Discard the flow-through.

• Add 200μL of wash buffer and spin 30 sec.

• Place siliva volumn into a new 1.5 ml tube. Add water directly to the column matrix and spin to elute the DNA.

Agarose gel electrophoresis

Digestion

PCR

Gel extract

Dephosphorylation of DNA

Phosphorylation of DNA

Ligation

Time Delay Test