IGEM:MIT/2007/Notebook/2007-8-11

Agenda

 * 1) Double digestion:
 * 2) *R0051.B0034, I14032.B0034, CPX.B0014 - for 3A assembly into pSB1AC3.
 * 3) *F2620.B0034.CPX, B0014 - for standard assembly and to figure out extra bands in western blot
 * 4) Purify digestion reactions. Remember to use BUFFER TE and NOT H2O.
 * 5) Ligations of digestion mixtures
 * 6) *R0051.B0034.CPX.B0014
 * 7) *I14032.B0034.CPX.B0014
 * 8) *F2620.B0034.CPX.B0014
 * 9) Transform ligations into DH5a and plate.

DD Mixtures
4.88 µL F2620.B0034.CPX (cPCR#6 205.0 ng/µL) 1 µL    SpeI 1 µL    PstI 0.5 µL  BSA 5 µL    NEB Buffer 2 37.62   µL H20 _______________ 50 µL Total

4.57 µL F2620.B0034.CPX (cPCR#8 218.7 ng/µL) 1 µL    SpeI 1 µL    PstI 0.5 µL  BSA 5 µL    NEB Buffer 2 37.93   µL H20 _______________ 50 µL Total

16 µL   B0014 (62.8ng/µL) 1 µL    XBaI 1 µL    PstI 0.5 µL  BSA 5 µL    NEB 3 26.5 µL H20 _______________ 50 µL Total

Digestion Nanodrops

 * F2620.B0034.CPX.cPCR6: 14.9 ng/µl
 * F2620.B0034.CPX.cPCR8: 16.3 ng/µl
 * B0014: 10.9 ng/µl

Ligation
3.5 µl    dd F2620.B0034.CPX.cPCR6 1 µl      dd B0014 2 µl      10x T4 Ligase Buffer 0.5 µl    T4 Ligase 13 µl     H20

20 µl      Total

3.5 µl    dd F2620.B0034.CPX.cPCR8 1 µl    dd B0014 2 µl    10x T4 Ligase Buffer 0.5 µl    T4 Ligase 13 µl    H20

20 µl      Total

3.5 µl     dd F2620.B0034.CPX.cPCR6 2 µl       10x T4 Ligase Buffer 0.5 µl     T4 Ligase 14 µl      H20

20 µl      Total

3.5 µl    dd F2620.B0034.CPX.cPCR8 2 µl      10x T4 Ligase Buffer 0.5 µl    T4 Ligase 14 µl     H20

20 µl      Total

1 µl      dd B0014 2 µl      10x T4 Ligase Buffer 0.5 µl    T4 Ligase 16.5µl    H20

20 µl      Total