IGEM:MIT/2007/Notebook/2007-8-14

Sequencing of Standard Assembled Parts

• CPX+B0014: Sample 1: okay except single substitution towards end of CPX sequence, Samples 2-4 successful
• R0051+B0034: Samples 1&3 failed to sequence, Samples 2&4 had R0051 only
• I14032+B0034: Samples 1-4 successful

• Next step: 3A ligate I14032+B0034 with CPX+B0014 into pSB1AC3 - use both #4 samples

Double Digestion

8 µl       CPX+B0014 (Sample #4: 135.3 ng/5µl)
0.5 µl     BSA
5 µl       NEB3 Buffer
1 µl       XbaI
1 µl       PstI
34.5 µl    H2O
------------------
50 µl      Total


5 µl       I14032 and B0034 (Sample #4: 225.7 ng/µl)
0.5 µl     BSA
5 µl       NEB2 Buffer
1 µl       EcoRI
1 µl       SpeI
37.5 µl    H2O
------------------
50 µl      Total

8 µl       F2620+B0034 (Sample #3 from LC4(same as used for CPX construct: 135 ng/µl)
0.5 µl     BSA
5 µl       NEB3 Buffer
1 µl       EcoRI
1 µl       PstI
34.5 µl    H2O
------------------
50 µl      Total

DD Nanodrop Results

CPX.B0014 (X,P) - 9.9 ng/µL

I14032.B0034 (E,S) - 14.4 ng/µL

F2620.B0034 (E,P) - 8.8 ng/µL

Ligations - Take 2

1 µl   dd pSB1AC3 (53 ng/µl)
1 µl   dd I14032+B0034
6 µl   dd CPX+B0014
2 µl     10x T4 Ligase Buffer
0.5 µl   T4 Ligase
9.5 µl   H20
-------------------
20µl      Total

1 µl      dd pSB1AC3 (53 ng/µl)
0 µl      dd I14032+B0034
0 µl      dd CPX+B0014
2 µl      10x T4 Ligase Buffer
0.5 µl    T4 Ligase
16.5 µl   H20
-------------------
20µl      Total

0 µl      dd pSB1AC3 (53 ng/µl)
0 µl      dd I14032+B0034
6 µl      dd CPX+B0014
2 µl      10x T4 Ligase Buffer
0.5 µl    T4 Ligase
11.5 µl   H20
-------------------
20µl      Total

0 µl      dd pSB1AC3 (53 ng/µl)
1 µl      dd I14032+B0034
0 µl      dd CPX+B0014
2 µl      10x T4 Ligase Buffer
0.5 µl    T4 Ligase
16.5 µl   H20
-------------------
20µl      Total

25 µl     dd R0051
0.5 µl    dd B0014
3 µl      10x T4 Ligase Buffer
0.5 µl    T4 Ligase
1 µl      H20
-------------------
30µl      Total


25 µl     dd R0051
0 µl      dd B0014
3 µl      10x T4 Ligase Buffer
0.5 µl    T4 Ligase
1.5 µl    H20
-------------------
30µl      Total

1 µl      dd pSB1AC3  (53 ng/µl)
9 µl      dd F2620+B0034
2 µl      10x T4 Ligase Buffer
0.5 µl    T4 Ligase
7.5 µl    H20
-------------------
20µl      Total

0 µl      dd pSB1AC3  (53 ng/µl)
9 µl      dd F2620+B0034
2 µl      10x T4 Ligase Buffer
0.5 µl    T4 Ligase
8.5 µl    H20
-------------------
20µl      Total