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From 2012.igem.org

Week 2 (06.25-07.01): Amplify pCJDD0, pCJDFA and pCJDFB

June 25

Pick up single colony of pCJDD0 and pCJDFA from plate of June 22 and amplify them in liquid LB medium.

June 26

1. Transform pCJDD0 (Ampr), pCJDFA (Spr) and pCJDFB (Kanr) into DH5α and DH10β respectively and adjust the resistance from that of June 22 because of a small mistake in supplementary information of reference.

2. Miniprep plasmid of pCJDD0 from bacteria of June25.

Name: D0-6-26-1 Concentration: 27.5 ng/ul A260/280: 1.76

Name: D0-6-26-2 Concentration: 24.7 ng/ul A260/280: 1.81

3. Transform plasmid D0-6-26-2 into DH10β

June 27

1. Pick up single colony of plate of D0-6-26-2, pCJDFA (DH10β), pCJDFB (DH10β) of June 26 and amplify them in liquid LB medium for minipreparing plasmids later.

2. Miniprep plasmid of D0-6-26-2 from bacteria of June27.

Name: D0-6-27-1 Concentration: 3.0 ng/ul A260/280: 2.36

Name: D0-6-27-2 Concentration: 2.0 ng/ul A260/280: -8.61

Name: D0-6-27-3 Concentration: 3.6 ng/ul A260/280: 1.70

Name: D0-6-27-4 Concentration: 3.8 ng/ul A260/280: 1.73

We forget to add ethanol in Solution W2 of Axygen Miniprep Kit, leading the failure of the experiment.

June 28

1. Miniprep plasmid of D0-6-26-2 and pCJDFA from bacteria of June27.

Name: D0-6-28-1 Concentration: 16.2 ng/ul A260/280: 2.11

Name: D0-6-28-2 Concentration: 21.0 ng/ul A260/280: 1.80

[Sequencing]

Name: D0-6-28-3 Concentration: 21.2 ng/ul A260/280: 1.77

[Sequencing]

Name: FA-6-28-1 Concentration: 4.3 ng/ul A260/280: 1.79

Name: FA-6-28-2 Concentration: 4.9 ng/ul A260/280: 2.00

Name: FA-6-28-3 Concentration: 7.3 ng/ul A260/280: 1.64

2. Pick up single colony of plate of D0-6-26-2, pCJDFA (DH5α), pCJDFB (DH5α) of June 26 and amplify them in liquid LB medium for minipreparing plasmids later.

3. Restriction enzyme digestion of D0-6-28-1, D0-6-28-2 and D0-6-28-3 with EcoR1 and Pst1.

It seems that we haven't achieved the correct plasmid but this may be caused by low concentration.

June 29

Amplify pCJDFA (DH5α) from bacteria of June 28.

June 30

1. Miniprep plasmid of pCJDFA (DH5α) from bacteria of June 29.

Name: FA-6-29-1 Concentration: 12.5 ng/ul A260/280: 1.29 [Sequencing]

Name: FA-6-29-2 Concentration: 12.7 ng/ul A260/280: 1.49 [Sequencing]

Name: FA-6-29-3 Concentration: 7.2 ng/ul A260/280: 0.96

2. Amplify pCJDFB (DH5α) and pCJDFB (DH10β) from bacteria of June28.

July 01

1. Receive correct sequencing results of D0-6-28-2 and D0-6-28-3.

2. Amplify bacteria of D0-6-28-2 and D0-6-28-3 for glycerol stock.

3. Miniprep plasmid of pCJDFB from bacteria of June 30.

Name: FB-7-01-1 Concentration: 7.1 ng/ul A260/280: 2.20

Name: FB-7-01-2 Concentration: 25.8 ng/ul A260/280: 2.05

[Sequencing]

Name: FB-7-01-3 Concentration: 20.4 ng/ul A260/280: 2.03

Name: FB-7-01-4 Concentration: 18.0 ng/ul A260/280: 1.83

[Sequencing]