Difference between revisions of "Team:UCLA/Notebook/Recombinant Expression"
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<summary>05/13:</summary> | <summary>05/13:</summary> | ||
Starter culture grew. Inoculation method is as follows: | Starter culture grew. Inoculation method is as follows: | ||
| − | grew 2 150 mL cultures at 30 and 37C respectively. Added 5mL of starter culture to each. Added Chlor at 1000X dilution. Added IPTG right away at 0.5mM concentration. This is non ideal but worked with my time constraints. This also correlates with expression protocol I used in | + | grew 2 150 mL cultures at 30 and 37C respectively. Added 5mL of starter culture to each. Added Chlor at 1000X dilution. Added IPTG right away at 0.5mM concentration. This is non ideal but worked with my time constraints. This also correlates with expression protocol I used in Yeates' lab. Inducing right away decreases the time we need to be in lab. I will compare the effectiveness of this method to other methods in the coming weeks. I will also compare the diff growth temperatures with both methods. |
</detail> | </detail> | ||
<detail> Thursday | <detail> Thursday | ||
Revision as of 23:36, 15 May 2015
Recombinant Silk Functionalization
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mm/dd: Sample Entry
Today we began cloning our GFP.
- PCR'd off template
- Ran gel
- Restriction digest
- Ligated into backbone
For our ligation, we made the following modifications:
- Tried it with newly bought ligase
- Left reaction overnight instead of 2 hrs
- Vector to insert ratio was 1:5 instead of 1:3
PCR Reaction:
| Component | Volume |
| 5X Q5 Reaction Buffer | 5 |
| 10 mM dNTPs | 0.5 |
| 10 uM Forward (primer 3/7) | 1.25 |
| 10 uM Reverse (primer 8) | 1.25 |
| Template (diluted to 1ng/uL) | 0.5 |
| Q5 High Fidelity DNA Polymerase | 0.25 |
| Nuclease Free Water | 16.25 |
Gel: Lot of bands, all at correct sizes
Goals
Achievements
What to accomplish next
Raw lab notebook entries
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