IGEM:MIT/2007/Notebook/2007-8-11

From OpenWetWare

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