IGEM:MIT/2007/Notebook/2007-8-3

Took Out LC's of I14032, B0014, R0051

• 1ml for glycerol stock
• rest miniprepped (for sequencing and 3A ligation)
  • when spun down, B0014 cells slightly reddish tint? Also, did not pellet well in spin down after adding buffer N3. Sample discarded and new LC made with second batch of transformed cells.
  • Nanodrop results for other two: I14032(165.2 ng/µl), R0051(16.8 ng/µl)

Digestion of I14032, B0034

6.5 µl     I14032 (165.2 ng/µl)
0.5 µl     BSA
5 µl       NEB2 Buffer
1 µl       SpeI
1 µl       PstI
36 µl      H20
--------------------------
50 µl      Total

12 µl      B0034 (84.9 ng/µl)
0.5 µl     BSA
5 µl       NEB3 Buffer
1 µl       XbaI
1 µl       PstI
30.5 µl    H2O
--------------------------
50 µl      Total

5 µl         CPX (1µg/5µl)
0.5 µl       BSA
5 µl         EcoRI Buffer
1 µl         EcoRI
1 µl         SpeI
37.5 µl      H2O
--------------------------
50 µl        Total

Gel Electrophoresis of Digestions

Wells (top to bottom)

• 2-log, B0034, CPX, I14032, 1 kb

Expected lengths:

• I14032: 37bp insert, 2.7kbp backbone
• B0034: 12bp insert, 2kbp backbone
• CPX: 720 bp insert, 3.5kbp backbone

Transformations

• Transformed DH5 alpha with the mer operon and the gfp part to send for sequencing
• PCR mer operon in psb3k3
• I3500 in psb3k3

Figure 5: Digest Gel Image