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

Agarose gel electrophoresis

Digestion

PCR

Gel extract

Dephosphorylation of DNA

Phosphorylation of DNA

Ligation

Time Delay Test