IGEM:MIT/2007/Notebook/2007-8-11: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
No edit summary
 
(3 intermediate revisions by one other user not shown)
Line 9: Line 9:
#*F2620.B0034.CPX.B0014
#*F2620.B0034.CPX.B0014
#Transform ligations into DH5a and plate.
#Transform ligations into DH5a and plate.
===DD Mixtures===
<pre>
4.88 µL  F2620.B0034.CPX (cPCR#6 205.0 ng/µL)
1 µL    SpeI
1 µL    PstI
0.5 µL  BSA
5 µL    NEB Buffer 2
37.62    µL H20
_______________
50 µL Total
4.57 µL  F2620.B0034.CPX (cPCR#8 218.7 ng/µL)
1 µL    SpeI
1 µL    PstI
0.5 µL  BSA
5 µL    NEB Buffer 2
37.93    µL H20
_______________
50 µL Total
16 µL    B0014 (62.8ng/µL)
1 µL    XBaI
1 µL    PstI
0.5 µL  BSA
5 µL    NEB 3
26.5 µL  H20
_______________
50 µL Total
</pre>
===Digestion Nanodrops===
*F2620.B0034.CPX.cPCR6: 14.9 ng/µl
*F2620.B0034.CPX.cPCR8: 16.3 ng/µl
*B0014: 10.9 ng/µl
===Ligation===
<pre>
3.5 µl    dd F2620.B0034.CPX.cPCR6
1 µl      dd B0014
2 µl      10x T4 Ligase Buffer
0.5 µl    T4 Ligase
13 µl      H20
--------------------
20  µl      Total
</pre>
<pre>
3.5 µl    dd F2620.B0034.CPX.cPCR8
1 µl    dd B0014
2 µl    10x T4 Ligase Buffer
0.5 µl    T4 Ligase
13 µl    H20
--------------------
20  µl      Total
</pre>
<pre>
3.5 µl      dd F2620.B0034.CPX.cPCR6
2 µl        10x T4 Ligase Buffer
0.5 µl      T4 Ligase
14 µl      H20
--------------------
20  µl      Total
</pre>
<pre>
3.5 µl    dd F2620.B0034.CPX.cPCR8
2 µl      10x T4 Ligase Buffer
0.5 µl    T4 Ligase
14 µl      H20
--------------------
20  µl      Total
</pre>
<pre>
1 µl      dd B0014
2 µl      10x T4 Ligase Buffer
0.5 µl    T4 Ligase
16.5µl    H20
--------------------
20  µl      Total
</pre>


==Holding off on constitutive promoter digestion/purification/ligation/transformation until sequencing results come back==
==Holding off on constitutive promoter digestion/purification/ligation/transformation until sequencing results come back==

Latest revision as of 08:41, 16 August 2007

Agenda

  1. Double digestion:
    • R0051.B0034, I14032.B0034, CPX.B0014 - for 3A assembly into pSB1AC3.
    • F2620.B0034.CPX, B0014 - for standard assembly and to figure out extra bands in western blot
  2. Purify digestion reactions. Remember to use BUFFER TE and NOT H2O.
  3. Ligations of digestion mixtures
    • R0051.B0034.CPX.B0014
    • I14032.B0034.CPX.B0014
    • F2620.B0034.CPX.B0014
  4. Transform ligations into DH5a and plate.

DD Mixtures


4.88 µL  F2620.B0034.CPX (cPCR#6 205.0 ng/µL)
1 µL     SpeI
1 µL     PstI
0.5 µL   BSA
5 µL     NEB Buffer 2
37.62    µL H20
_______________
50 µL Total


4.57 µL  F2620.B0034.CPX (cPCR#8 218.7 ng/µL)
1 µL     SpeI
1 µL     PstI
0.5 µL   BSA
5 µL     NEB Buffer 2
37.93    µL H20
_______________
50 µL Total


16 µL    B0014 (62.8ng/µL)
1 µL     XBaI
1 µL     PstI
0.5 µL   BSA
5 µL     NEB 3
26.5 µL  H20
_______________
50 µL Total

Digestion Nanodrops

  • F2620.B0034.CPX.cPCR6: 14.9 ng/µl
  • F2620.B0034.CPX.cPCR8: 16.3 ng/µl
  • B0014: 10.9 ng/µl

Ligation

3.5 µl     dd F2620.B0034.CPX.cPCR6
1 µl       dd B0014
2 µl       10x T4 Ligase Buffer
0.5 µl     T4 Ligase
13 µl      H20
--------------------
20  µl      Total

3.5 µl     dd F2620.B0034.CPX.cPCR8
1 µl     dd B0014
2 µl     10x T4 Ligase Buffer
0.5 µl     T4 Ligase
13 µl     H20
--------------------
20  µl      Total

3.5 µl      dd F2620.B0034.CPX.cPCR6
2 µl        10x T4 Ligase Buffer
0.5 µl      T4 Ligase
14 µl       H20
--------------------
20  µl      Total

3.5 µl     dd F2620.B0034.CPX.cPCR8
2 µl       10x T4 Ligase Buffer
0.5 µl     T4 Ligase
14 µl      H20
--------------------
20  µl      Total

1 µl       dd B0014
2 µl       10x T4 Ligase Buffer
0.5 µl     T4 Ligase
16.5µl     H20
--------------------
20  µl      Total

Holding off on constitutive promoter digestion/purification/ligation/transformation until sequencing results come back

Retrieved from "https://openwetware.org/mediawiki/index.php?title=IGEM:MIT/2007/Notebook/2007-8-11&oldid=141864"