IGEM:Harvard/2008/Lab Notebooks/DailyBook/Week12/Chemical and Light

9/22 RE digests

With NEB enzymes, 60 min (buffer 2+BSA):

• 128#1 XPXmnI
• 128#5 XSXmnI
• 128#9 ESXmnI

The XmnI is to cut the backbone, which has a similar length to the mtrB constructs.

With Fermentas enzymes, 25 min:

• 116 SP
• 117 SP
• 108 SP
• 51 EX
• 63 XP

9/23 ligations

The following ligations were performed using 1:6 molar ratio and the Takara kit at 16°C for 45min:

• 128ES+63EX (also did a 5min, 25°C ligation to test whether the extraction method or the ligation conditions are improving the process)
• 128XP+108SP
• 128XP+116SP
• 128XP+117SP
• 51EX+38ES
  • Neg ctrl with water as insert
• 117SP+128XS+[63XP]
• 108SP+128XS+[63XP]
• 116SP+128XS+[63XP]

Note that [63] means P63 was added 20 minutes into the ligation (to reduce uptake only of terminator). Prior to addition of ligase, DNA mix was heated to 65°C for 2 minutes and allowed to cool. For ones with 63 added later, the 63 was heated prior to addition, and more ligation mix was added next to make up the volume.

All transformed into DH5α.