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From 2012.igem.org

Week 7 (07.30-08.05): Test part BBa_K537009 and make our own parts

July 30

1. Miniprep plasmid of K738000-7-29.

Name: K738000-7-30-1 Concentration: 60.0 ng/ul A260/280: 1.86

Name: K738000-7-30-2 Concentration: 47.1 ng/ul A260/280: 1.95

Name: K738000-7-30-3 Concentration: 90.1 ng/ul A260/280: 1.95

Name: K738000-7-30-10 Concentration: 43.6 ng/ul A260/280: 1.98

PCR of K738000-7-30

Unfortunately, there was a slight electrophoretic band in control. But the sequencing result came later verified that we've got the correct ligation product.

2. Pick up single colonies from plate of FB-7-26-2 and amplify it in LB liquid medium.

July 31

1. Amplify backbone pSB1C3:

Double digest K738000-7-23-3 with EcoR1 and Pst1 and got linear backbone through DNA gel extraction.

2. Miniprep plasmid of K738000-7-29 and FA-7-28-1.

Name: K738000-7-30-4 Concentration: 83.9 ng/ul A260/280: 1.94

Name: K738000-7-30-5 Concentration: 50.1 ng/ul A260/280: 2.00

Name: K738000-7-30-11 Concentration: 46.0 ng/ul A260/280: 1.97

Name: FA-7-28-1 Concentration: 33.0 ng/ul A260/280: 2.04

August 01

Double digest K738000-7-23-2 and K738000-7-23-3 with EcoR1 and Pst1 and got linear backbone through DNA gel extraction.

August 02

1. Transform part K537009 into DH5α to acquire experience, named venus-8-2.

2. Transform original pCJDFA and pCJDFB into DH5α, named FA-8-2 and FB-8-2.

3. Transform part K398326 into DH10β to amplify backbone pSB1C3 easier, BB-8-2.

August 03

1. Amplify BB-8-2, FA-8-2 and FB -8-2 for minipreparing plasmids later, named BB-8-3, FA-8-3 and FB-8-3.

August 04

1. Miniprep plasmid of BB-8-3 FA-8-3 and FB-8-3.

Name: BB-8-4 Concentration: 76.2 ng/ul A260/280: 1.91

Name: BB-8-4 Concentration: 40.3 ng/ul A260/280: 1.90

Name: FA-8-4 Concentration: 19.6 ng/ul A260/280: 1.97

Name: FB-8-4 Concentration: 35.9 ng/ul A260/280: 1.98

Unfortunately, the PCR results were so different from that of original plasmids. We couldn't figure out the reason.

2. Amplify Venus-8-2 in LB liquid medium, named Venus-8-4.

3. Transform FAM-8-4-1, FAM-8-4-1, FBP-8-4-1 and FBP-8-4-1 into DH5α.

August 05

1. Pick up single colonies from plate of FAM-8-4 and FBP-8-4 and amplify it in LB liquid medium and LB plates.

2. Colony PCR of FAM-8-4-2 and FBP-8-4-2

3. Double digest BB-8-4-1 with (1) EcoR1 and Pst1, (2) Xba1 and Spe1, get linear backbone through PCR cleanup kit.

4. Miniprep plasmid of Venus-8-4.

Name: Venus-8-5-1 Concentration: 168.1 ng/ul A260/280: 1.91

Name: Venus-8-5-1 Concentration: 83.4 ng/ul A260/280: 1.93

5. Pick up single colonies from plate of FAM-8-4-1, FAM-8-4-2, FBP-8-4-1 and FBP-8-4-2 and amplify it in LB liquid medium and LB plates. Miniprep plasmid of them.

Name: FAM-8-5-1 Concentration: 69.9 ng/ul A260/280: 1.95

Name: FAM-8-5-2 Concentration: 57.3 ng/ul A260/280: 1.95

Name: FAM-8-5-3 Concentration: 86.8 ng/ul A260/280: 2.06

Name: FBP-8-5-2 Concentration: 21.0 ng/ul A260/280: 2.14

Name: FBP-8-5-3 Concentration: 15.5 ng/ul A260/280: 2.14

Name: FBP-8-5-5 Concentration: 15.6 ng/ul A260/280: 2.07

6. Co-transform FA-8-5-3, FB-8-5-2 and D0-4 into BL21*(DE3). Co-transform FA-8-5-3 and FB-8-5-2 into BL21*(DE3).