Team:UCLA/Notebook/Spider Silk Genetics/25 August 2015
iGEM UCLA
8/25/2015
Transformation Results
- M1-12, M1/2[1:1]-12(1C3)/(T7), M1/2[2:1]-12(1C3)/(T7) all have several dozens of colonies.
PCR amplification for M1/2[1:2]-12
|
|
Volume (uL)
|
| 5x Q5 Buffer
|
10
|
| 10 mM dNTPs
|
1
|
| 10 uM For (F-03)
|
2.5
|
| 10 uM Rev (G-03)
|
2.5
|
| Template
|
1
|
| Q5 Polymerase
|
0.5
|
| ddH2O
|
32.5
|
| Total
|
50 uL
|
| 98 C
|
30 sec
|
| 98 C
|
10 sec
|
| 66 C
|
20 sec
|
| 72 C
|
30 sec
|
| repeat from step 2
|
20x
|
| 72 C
|
2 min
|
| 12 C
|
hold
|
- Visualized on 1% TAE gel, with 2 uL of NEB 1 kb ladder.
- Excised gel slice corresponding to 12-mer size (1324 bp), and gel extracted DNA.
RE Digestion
- Digest all of M1/2[1:2]-12 in a 50 uL reaction with EcoRI, PstI.
|
|
Volume (uL)
|
| NEBuffer 2.1
|
5
|
| DNA
|
20
|
| ddH2O
|
23
|
| EcoRI
|
1
|
| PstI
|
1
|
| Total
|
50
|
- Digest at 37 C for 1.5 hrs, heat kill at 65 C for 20 min.
- PCR purified digestion:
Ligation
- Ligated into pSB1C3 in 20 uL reaction using T4 Ligase.
- Ligated at 25 C for 1 hr, heat kill at 65 C for 20 min.
Transformation
- Dialyzed 3 uL of ligation product against ddH2O.
- Transformed 2 uL of ligation into electrocompetent DH5(alpha) cells.
Liquid Culture
- Picked three colonies each from the plates this morning into 5 mL of LB + chlor.
- 15 colonies total.
