Difference between revisions of "Team:UCLA/Notebook/Honeybee Silk/10 July 2015"
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| − | Took | + | *Took it out at 9:30 am 7/11 |
Revision as of 17:16, 11 July 2015
Double Digestion and Cleanup of pET24a
- Sequencing results checked out for miniprepped pet 24a plasmid.
- I will be using the first sample from 7/9 (OD: 231.26ng/uL)
Digestion Reaction
| Component | Volume (out of 50uL) |
|---|---|
| NEB 2.1 Buffer | 5uL |
| BamHI | 1uL |
| NdeI | 1uL |
| pET24a (Miniprepped) | 5uL |
| Nuclease Free Water | 38uL |
Program
| Step | Temperature | Time |
|---|---|---|
| Incubation | 37C | 1 hour |
| Heat Inactivation | 80C | 15 min |
| Hold | 10C | Hold |
I ran the sample on a 1% agarose gel against a 1kB ladder. The appropriate band length was present (5310 bp).
(picture, do this asap)
The band was excised from the gel and purified along with the collected gel sample of the honeybee insert from 7/8 using the Qiagen Gel Purification kit.
OD: Honeybee silk: 28.4ng/uL pET24a: 23.6
Ligation
- Ran ligation reaction with double digested pet 24a and honeybee sequenced
| Component 3:1 insert:vector) | Volume (out of 20uL) |
|---|---|
| T4 Ligase Buffer | 2 ul |
| Pet 24a backbone (23.6 ng/ul) | 2.54 ul |
| SIlk insert 28.4 ng/ul | 1.12 |
| T4 Ligase | 1 ul |
| Water | 13.34 |
| Component 5:1 insert to vector | Volume (out of 20uL) |
|---|---|
| T4 Ligase Buffer | 2 ul |
| Pet 24a backbone (23.6 ng/ul) | 2.54 ul |
| SIlk insert 28.4 ng/ul | 1.9 |
| T4 Ligase | 1 ul |
| Water | 12.56 |
- Running ligation reaction overnight 16 C starting at 6:45 pm 7/10
- Took it out at 9:30 am 7/11