Difference between revisions of "Team:UCLA/Notebook/Protein Cages/7 August 2015"

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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.
 
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.
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Intro: Performed a 50 uL PCR amplification of Mutant #10. PCR cleanup.
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50 uL PCR:
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10 uL 5x Q5 buffer
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5 uL 2mM dNTPs
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2.5 uL 10 uM forward primer
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2.5 uL 10 uM reverse primer
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0.5 uL 1 ng/uL template DNA
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0.5 uL Q5 Polymerase
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29 uL ddH2O (to 50 uL)
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This was mixed in a master tube and added to two different PCR tubes.
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98C 30s
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98C 10s }
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Tm  15s } 25x
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72C 30s }
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72C 5min
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PCR Cleanup: Performed according to recommended instructions.

Revision as of 20:45, 19 August 2015

iGEM UCLA




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.