Difference between revisions of "Team:UCLA/Notebook/Honeybee Silk/10 July 2015"
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I ran the sample on a 1% agarose gel against a 1kB ladder. The appropriate band length was present (5310 bp). | I ran the sample on a 1% agarose gel against a 1kB ladder. The appropriate band length was present (5310 bp). | ||
| − | + | [[File:Gel 2015-07-10.jpg|none|thumb|500px|Gel Image]] | |
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. | 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. | ||
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pET24a: 23.6 | pET24a: 23.6 | ||
| − | ='''Ligation'''= | + | =='''Ligation'''== |
*Ran ligation reaction with double digested pet 24a and honeybee sequenced | *Ran ligation reaction with double digested pet 24a and honeybee sequenced | ||
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| − | Running ligation reaction overnight 16 C starting at 6:45 pm 7/10 | + | *Running ligation reaction overnight 16 C starting at 6:45 pm 7/10 |
| + | *Took it out at 9:30 am 7/11 | ||
Latest revision as of 20:37, 15 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).
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