Team:UCLA/Notebook/Spider Silk Genetics/29 July 2015
iGEM UCLA
7/29/2015
Sequencing Results for M2-12(1C3)
- 1: Bad: No reverse priming site.
- 2: long deletion in beginning.
- 3: VF: good from beginning to 777. VR: good from 510 to end.
Gel Purification for M1-AB, BC, CA; M2-15; linear pSB1C3
- Used Zymo Gel Recovery Kit.
|
|
Concentration (ng/uL)
|
A 260/280
|
| M1-AB
|
24.64
|
2.08
|
| M1-BC
|
31.85
|
1.95
|
| M1-CA
|
30.79
|
1.71
|
| M2-15
|
15.75
|
1.41
|
| pSB1C3
|
319.11
|
1.83
|
Digestion for M2-12(1C3), M2-15, pSB1C3
- Digested each in 50 uL reactions, buffered with NEBuffer 2.1
- Used 1 uL for each of the indicated enzymes.
|
|
Digestion Amount (ug)
|
Enzymes
|
Post-Digestion Purification
|
Post-Digestion Concentration (ng/uL)
|
| M2-12
|
3
|
XbaI, PstI
|
Gel Purification
|
71.06
|
| M2-15
|
0.375
|
EcoRI, PstI
|
PCR Purify
|
16.93
|
| pSB1C3
|
1 ug
|
EcoRi, PstI
|
PCR Purify
|
65.85
|
Ligation for M2-12 into BBa_K525998, M2-15 into pSB1C3
- Used ligation calculator.
- Vector size: 2070 bp
- Vector amount: 50 ng
- Insert size: 1324 (M2-12), 1630 (M2-15)
- Vector to insert ratio: 1:3
- Ligate:
- 95.9 ng of M2-12 to 50 ng of vector.
- 118.1 ng of M2-15 to 50 ng of vector.
- Ligate for 1 hr at 25 C, heat kill at 65 C for 15 min.
Transformation
- M2-12(T7) into BL21(DE3) chemically competent cells.
- M2-15(1C3) into DH5(alpha) electrically competent cells.
- Dialyzed and transformed 2 uL of DNA.
- Arc time: 5.5 ms
