Team:UCLA/Notebook/Spider Silk Genetics/15 July 2015
iGEM UCLA
7/15/2015
Sequence Verification
- ICA MaSp2 3-mer
- MaSp2 6-mer
- Sequence failure for 1, 2.
- Sequence 3 is good.
- MaSp2 Seq AB
- 1: BAD: single bp deletion
- 2: GOOD
- 3: BAD: 20 bp insert
Restriction Enzyme Digestion, Subcloning Preparation
- Subcloning 3-mer and 6-mer into BBa_K525998 for expression.
- Cloning 9-mer into pSB1C3 for sequencing and biobrick making.
|
|
Restriction Enzymes
|
Amount to Digest
|
Post-Digest Processing
|
| BBa_K525998
|
S, P
|
1 ug of plasmid
|
PCR purify
|
| 3-mer
|
X, P
|
2 ug of plasmid
|
Gel purify
|
| 6-mer
|
X, P
|
2 ug of plasmid
|
Gel purify
|
| 9-mer
|
E, P
|
10 uL of solution
|
PCR purify
|
- Digestion was in 50 uL reactions; all used 10x NEBuffer 2.1; used 1 uL of each enzyme.
- Digest at 37 C for 1.5 hrs, then heat kill at 65 C for 20 min.
Results
|
|
Concentration (ng/uL)
|
A 260/280
|
| BBa_K525998
|
102.46
|
2.07
|
| MaSp2 3-mer
|
32.33
|
2.53
|
| MaSp2 6-mer
|
31.82
|
1.70
|
| MaSp2 9-mer
|
10.28
|
.1.73
|
Ligation for Transformation Preparation
- Ligate MaSp2 3-mer, 6-mer into BBa_K525998.
- Ligate MaSp2 ICA 9-mer into pSB1C3.
- Used [http://www.insilico.uni-duesseldorf.de/Lig_Input.html Ligation Calculator] with the following parameters to determine relative DNA amounts:
- vector size: 2200 bp
- vector amount: 50 ng
- insert size:
- 3-mer: 406 bp
- 6-mer: 712 bp
- 9-mer: 1018 bp
- 3:1 ratio of insert:vector
- Ligation in 20 uL reactions with 1 uL of T4 Ligase.
- Ligated at 25 C for 25 min, then heat kill at 65 C for 10 min.
Transformation
- 2 uL of ligated product MaSp2 3-mer, 6-mer were transformed into BL21(DE3) chemical competent cells.
- Plated all the bacteria onto chloramphenicol plates.
- 3 uL of ligated MaSp2 ICA 9-mer were dialyzed against ultra-pure water.
- Transformed 2 uL of MaSp2 ICA 9-mer into DH5(alpha) electrocompetent cells.
- Plated all the bacteria onto chloramphenicol plates.
