Difference between revisions of "Team:UCLA/Notebook/Protein Cages/7 August 2015"
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Intro: Performed a 50 uL PCR amplification of Mutant #10. PCR cleanup. | Intro: Performed a 50 uL PCR amplification of Mutant #10. PCR cleanup. | ||
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50 uL PCR: | 50 uL PCR: | ||
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PCR Cleanup: Performed according to recommended instructions. | PCR Cleanup: Performed according to recommended instructions. | ||
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Latest revision as of 20:46, 19 August 2015
Introduction: Today gel extraction and digestion of PCquad 3.0 will be done. If permitting, ligation will also be done.
Procedures: Gel extraction was performed as indicated by zymo procedures. The concentration was measured using the nanodrop. 78.78ng/uL. 260/280 = 3.23.
Since the yield was too low, another round of amplification will need to be done before digestion.
Conclusions: There weren’t any restriction enzymes available to use, so digestion will be done next week. PCR amplification will be done in a larger volume for PCquad 3.0 as well.
Intro: Performed a 50 uL PCR amplification of Mutant #10. PCR cleanup.
50 uL PCR:
10 uL 5x Q5 buffer
5 uL 2mM dNTPs
2.5 uL 10 uM forward primer
2.5 uL 10 uM reverse primer
0.5 uL 1 ng/uL template DNA
0.5 uL Q5 Polymerase
29 uL ddH2O (to 50 uL)
This was mixed in a master tube and added to two different PCR tubes.
98C 30s
98C 10s }
Tm 15s } 25x
72C 30s }
72C 5min
PCR Cleanup: Performed according to recommended instructions.
Tyler Lee