IGEM:MIT/2007/Notebook/2007-8-11
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Agenda
- 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
- Purify digestion reactions. Remember to use BUFFER TE and NOT H2O.
- Ligations of digestion mixtures
- R0051.B0034.CPX.B0014
- I14032.B0034.CPX.B0014
- F2620.B0034.CPX.B0014
- 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