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