Binding Site Design - October

02/10/2010

• digestion in 50 µl of
  • 4x pSB1C3 (5µl BSA, 5µl NEB buffer 2, 1µl PstI, 1µl EcoRI, 1µl DpnI, 4µl construct, 33 µl H2O)
  • 4x pSB1A3 (5µl BSA, 5µl NEB buffer 2, 1µl PstI, 1µl EcoRI, 1µl DpnI, 4µl construct, 33 µl H2O)

05/10/2010

• 1µg digestion of miMeasure and 6 different constructs (1A, 3F, 13, 15, 17, 37) with EcoRI and PstI

1= 0S

2= 0L

3= 10S

4= 10L

06/10/2010

20 Miniprep:

• digestion of miMeasure and 6 different synthetic miRNA patterns against endogenous miR122
• gel seperation
• gel purification
• sequencing

12/10/2010

4 miraPCRs for binding sites of miR4286.

• one PCR with an annealing temperature of 45 degree and 16 cycles
• second miraPCR with annealing temperature of 57 degree and also 16 cycles
  • nucleotide removal
  • digestion
  • gel seperation
  • gel purification
  • ligation in miMeasure and pSB1C3
  • transformation

13/10/2010

• pick colonies
• about 8 hours later: do minipreps
• send for sequencing