Difference between revisions of "Team:UCLA/Notebook/Spider Silk Genetics/19 August 2015"
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[[File:8 19 2015 UCLA ICA.jpg|none|thumb|500px|'''Fig. 1'''PCR amplification of M1/2[1:1]-12 ICA eluate. Both lanes are identical reactions. The expected size for 12-mer amplification is 1324 bp. The most prominent band in both reactions is a ~1.3 kb band. There are accessory bands corresponding to 3-, 6-, 9-mers as well.]] | [[File:8 19 2015 UCLA ICA.jpg|none|thumb|500px|'''Fig. 1'''PCR amplification of M1/2[1:1]-12 ICA eluate. Both lanes are identical reactions. The expected size for 12-mer amplification is 1324 bp. The most prominent band in both reactions is a ~1.3 kb band. There are accessory bands corresponding to 3-, 6-, 9-mers as well.]] | ||
*This result indicates that ICA for M1/2[1:1]-12 was successful. | *This result indicates that ICA for M1/2[1:1]-12 was successful. | ||
+ | |||
+ | ==Large Scale Liquid Culture for M2-AB, BC, CA== | ||
+ | *100 mL of each. Picked a single colony from freshly streaked plates. Used the first version of M2 monomers for this. |
Revision as of 05:20, 24 August 2015
Contents
8/19/2015
Sequencing Results
- M1-12(1C3):
- 1: BAD (VR is bad; no priming)
- 2: good
- 3: good
- 4: good?
- Further inspection reveals BBa_K525998 T7-RBS is present in the beginning. The sequence is otherwise correct. This is suspicious, and we will do a Restriction test to verify proper length.
- ERRATA: All these sequences, while correct, are not the appropriate size. These are in fact, M1-9(1C3) and M1-9(T7) plasmids.
ICA for M1/2[1:1]-12
- Followed previous ICA protocol using:
- 45 minute initiator binding time.
- 3 minute ligation times for each step.
- Elute in 15 uL of 0.01% Tween-20 at 95 C for 5 min.
PCR Amplification for M1/2[1:1]-12
- 2x 50 uL reactions
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 (ICA eluate) | 1 |
ddH2O | 32.5 |
Q5 Polymerase | 0.5 |
Total | 50 |
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 |
Results
- Cast 1% TAE gel to visualize. Used 2 uL of NEB 1 kb ladder.
- This result indicates that ICA for M1/2[1:1]-12 was successful.
Large Scale Liquid Culture for M2-AB, BC, CA
- 100 mL of each. Picked a single colony from freshly streaked plates. Used the first version of M2 monomers for this.