Team:Xiamen City/Notebook



a) Safety training

b) Study basic techniques used during experiments, and the PCR technology

c) Prepare culture medium (YPD medium and LB medium).

d) Transfer Cas9 and gRNA plasmid DNA into two groups of Escherichia coli.

e) Apply PCR to the upstream and downstream homology arms.


f) Learn how to test OD

g) Learn the electrophoresis technology

h) Proliferate the two groups of Escherichia coli.

i) Transform the yeast cells with Cas9 plasmid DNA into competent state.

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1: pgaA PCR product

2: pgaA PCR product

3: HR-L PCR product

4: HR-R PCR product


j) Learn the column extraction method

k) Extract Cas9 and gRNA plasmid DNA from the two groups of Escherichia coli.

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1: pHCas9-Nourse-1

2: pHCas9-Nourse-2

3: pYES2-gRNA-hyg-MCS-1

4: pYES2-gRNA-hyg-MCS-2

5: HR-L PCR product

6: HR-L PCR Product


l) Prepare the double antibiotics YPD medium.

m) Cultivate the yeast with single fungus containing cas9 plasmid

n) Apply PCR to the pgaA sample.


o) Apply PCR to integrate the upstream, downstream homology template, and pgaA.

p) Cultivate yeast cells that contains Cas9 plasmid.

q) Rebuild 3 PCR samples (up+pgaA, pgaA+down, up+pgaA+down)


r) Examine previous PCR results.

s) Extract homology repair template.

t) Transfer the gRNA plasmid and homology repair template into the yeast competent cell containing the Cas9 plasmid.

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u) Prepare Congo red solution.

v) Prepare NaOH solution

w) Prepare NaCl solution


x) Prepare pectin medium for function test


y) Test the yeast solution at different concentration


z) Extract plasmid and send out for sequencing


aa) Sequencing feedback received


bb) Induction experiments

cc) DNS Test