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 Bold text