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

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(Ligations - Take 2)
Line 34: Line 34:
------------------
------------------
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 11:46, 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
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