IGEM:MIT/2007/Notebook/2007-8-14: Difference between revisions
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50 µl Total | 50 µl Total | ||
</pre> | |||
===Ligations - Take 2=== | |||
<pre> | |||
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 | |||
</pre> | </pre> |
Revision as of 08:46, 16 August 2007
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
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