Team:HokkaidoU Japan/Notebook/September16

• digestion of GFP(1-14K) and double terminator(1-23L)

Digestion of GFP and Double Terminator

Parts Information

1-14K and pSB1A3 had purified with mycrocon, and 1-23L had extracted from a gel before.

Method

1. electrophoresed 1ul of 1-14K and 1-23L added 0.5ul of 6×sample buffer to estimate concentration of each solution.
2. estimated concentration from photo of electrophoresys.But I forgot to electrophorese pSB1A3 solution with the other samples,so pSB1A3 solution was done by other person.
3. made digestion recipes based on each concentrations.Why pSB1A3 recipe is two,because I firstly made 30ul of pSB1A3 solution,but I found it was insufficient to ligate parts,so I made more 50ul of it after.

Estimates of concentration

1-14K:200ng/ul

1-23L:120ng/ul

pSB1A3:2.5ng/ul

Digestion Menu

1. put each solutions into 37℃ incubator.1-23L solution was put about a half and two hours,1-14K solution was done about a half and an hour,30ul of pSB1A3 solution was done about an hour,and 50ul of pSB1A3 was done about a half hour.

1. electrophoresed each solutions added 6×sample buffer.put 12uls each into wells of a gel.

• cannot see 1-23L because of overflowing.extracted the other samples from a gel.dissolve them with 50ul of Nuclease free water.And they were stocked to freeze in -20℃.