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

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(Ligations - Take 2)
(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 11:45, 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
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