Difference between revisions of "Team:UCLA/Notebook/Spider Silk Genetics/25 August 2015"
Vinsonclam (Talk | contribs) (Created page with "{{Template:UCLA}} =8/25/2015= ==Transformation Results== *M1-12, M1/2[1:1]-12(1C3)/(T7), M1/2[2:1]-12(1C3)/(T7) all have several dozens of colonies. ==PCR amplification for M1...") |
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Latest revision as of 04:33, 6 September 2015
Contents
8/25/2015
Transformation Results
- M1-12, M1/2[1:1]-12(1C3)/(T7), M1/2[2:1]-12(1C3)/(T7) all have several dozens of colonies.
PCR amplification for M1/2[1:2]-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 | 1 |
Q5 Polymerase | 0.5 |
ddH2O | 32.5 |
Total | 50 uL |
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 |
- Visualized on 1% TAE gel, with 2 uL of NEB 1 kb ladder.
- Gel image lost.
- Excised gel slice corresponding to 12-mer size (1324 bp), and gel extracted DNA.
- 49.26 ng/uL
RE Digestion
- Digest all of M1/2[1:2]-12 in a 50 uL reaction with EcoRI, PstI.
Volume (uL) | |
---|---|
NEBuffer 2.1 | 5 |
DNA | 20 |
ddH2O | 23 |
EcoRI | 1 |
PstI | 1 |
Total | 50 |
- Digest at 37 C for 1.5 hrs, heat kill at 65 C for 20 min.
- PCR purified digestion:
- 29.37 ng/uL
Ligation
- Ligated into pSB1C3 in 20 uL reaction using T4 Ligase.
- Ligated at 25 C for 1 hr, heat kill at 65 C for 20 min.
Transformation
- Dialyzed 3 uL of ligation product against ddH2O.
- Transformed 2 uL of ligation into electrocompetent DH5(alpha) cells.
- Arc time: 4.8 ms.
Liquid Culture
- Picked three colonies each from the plates this morning into 5 mL of LB + chlor.
- 15 colonies total.