Week 5 (07.16-07.22): Struggle to acquire correct plasmids

July 16

1. Miniprep plasmid of FA and FB from bacteria of July 12.

Name: D0-7-16-1 Concentration: 60.9 ng/ul A260/280: 1.65

Name: D0-7-16-2 Concentration: 34.0 ng/ul A260/280: 1.93

Name: Penta Concentration: 74.9 ng/ul A260/280: 1.91

Name: RFP Concentration: 201.2 ng/ul A260/280: 1.90

2. Amplify glycerol stock D0 bacteria on two Amp plates, named D0-7-16-1 and D0-7-16-2.

3. Identify FA-7-13-2-2, FA-7-13-2-3, FB-7-14-2 and FB-7-14-4 with PCR, new primers.

The result of FA is not very ideal, while that of FB is not very exactly. The plasmid (all was Miniprep today) seems just stay where they were put in.

July 17

1. Amplify FA-FB-7-15-90 and FA-FB-7-15-30 in LB liquid medium.

2. Identify FA-7-13-2-2, FA-7-13-3-1 with PCR, increase Tm by 2Degrees Celsius.

3. Amplify glycerol stock co-transformation-2-1 bacteria on plate, named co3-7-17.

July 18

Amplify FA-7-13-2-2, FA-7-13-2-3, FB-7-13-2 and FB-7-13-5 for minipreparing plasmids later.

July 19

1. Co-transformation of D0-7-16-2, FA-7-13-1-1 and FB-7-14-6, named Co3-7-18.

2. Amplify FB-7-12-1 and FB-7-12-3 in LB liquid medium.

3. Miniprep plasmid of FA-7-18-2-2 and FA-7-18-2-3.

Name: FA-7-18-2-2 Concentration: 17.4 ng/ul A260/280: 2.19

Name: FA-7-18-2-3 Concentration: 14.7 ng/ul A260/280: 2.27

July 20

Amplify glycerol stock Co3-2-1 bacteria on plate, named Co3-7-20.

July 21

1. Gradient PCR to find an appropriate Tm value for FA.

It seems that there is not a lot of differences.

2. Amplify Co3-7-20 on plates, named Co3-7-21.

3. Amplify the following bacteria:

No Name Form resistance plate

1 Co2-7-21-1 Co2-7-17-1 K+S K+S

2 Co2-7-21-2 Co2-7-17-2 K+S K+S

3 Co2-7-21-3 Co2-7-17-3 K+S K+S

4 control-kan DH10β - Kan

5 control-sp DH10β - Sp

6 control DH10β - -

July 22

1. Amplify Co3-7-21 in LB liquid medium, named Co3-7-22.

2. Colony PCR to test Co2-7-21, Co3-7-22, and FB-7-19.

We found that we may use a wrong protocol of colony PCR.

3. Co-transform D0, FA and FB, named Co3-7-22. Co-transform FA and FB, named Co2-7-22.

4. Amplify FA-7-18-2-2, FA-7-18-2-3, FB-7-18-2 for minipreparing plasmids later.

5. Amplify D0-7-15-2 and Co3-7-21 on other plate, named D0-7-22-2 and Co3-7-22.