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From 2012.igem.org

Week 11 Recuperate

2012/8/27 Monday, August 27, 2012

1. Chen did the overlap PCR and construct FB-2X-PP7 (approximately 630 bp). It seems correct in the agarose gel electrophoresis. (1000 marker on the left and 5000 marker on the right)

2. Yan and Chen PCR MS2 from plasmids Dr. Delebecque sent us respectively. Yan uses 57.5 Degrees Celsius as melting temperature and Chen uses 58.5 and 63.5. But the electrophoretic bands seem incorrect. (Next day we find angrily that the primer the company synthesized is wrong!)

2012/8/28 Tuesday, August 28, 2012

1. LIU uses gel extraction to get last day Chen's MS2 and carry on overlap PCR to make FA-2X-MS2.

2. Chen transforms FB-2X-PP7 (T vector) into Top 10.

3. Repeated tests: Yan does the same as LIU and sets annealing temperature 59Degrees Celsius and 61Degrees Celsius.

2012/8/29 Wednesday, August 29, 2012

4. Teacher Hu Yuhua from Edinburgh University interviews us concerning team collaboration. She offers that if we have oral English barriers we could come to her.

5. LIU makes competent cell of DH10β

2012/8/30 Thursday, August 30, 2012

All of us attend short semester and did nothing about igem today.

2012/8/31 Friday, August 31, 2012

1. Sequencing result shows that FB-2X-PP7 we have constructed was correct in T vector. So LIU prepare some for Miniprep and glycerol freeze store.

2. Enzyme digestion and ligation: make FB-2X-PP7 from T vector into pColaDuet and transform it in DH5α.

September 01

1. DNA gel extraction of FB-PP7-1 and FB-PP7-2.

Name: FB-PP7-1    Concentration: 0.8 ng/ul    A260/280: 2.23

Name: FB-PP7-2    Concentration: 1.4 ng/ul    A260/280: 1.63

2. Make competent cells of E.coli BL21*(DE3) strain.

3. Miniprep plasmid of pCDFDuet-8-31-1 and pCDFDuet-8-31-2.

Name: pCDFDuet-9-1-1    Concentration: 84.68 ng/ul    A260/280: 1.90

Name: pCDFDuet-9-1-2    Concentration: 94.84 ng/ul    A260/280: 1.88

4. Ligation: FB-PP7-1/ FB-PP7-2 and pColaDuet, and then transform it into DH5a competent cells.

5. Acquire MS2 with PCR procedure. Overlap PCR of FA and MS2 together to acquire FA-2X-MS2.

September 02

1. DNA gel extraction of FA-MS2-9-2-1 and FA-MS2-2-2.

Name: FA-MS2-9-2-1    Concentration: 11.8 ng/ul    A260/280: 1.79

Name: FA-MS2-9-2-2    Concentration: 16.0 ng/ul    A260/280: 1.73

2. Add 'A' to the DNA gel extraction product.

3. Ligation of the product of adding 'A' and then transform the product into Top 10 competent cells.

4. Pick up single colonies from plate of pCola-FB-PP7-9-1 and amplify them in LB liquid medium. Colony PCR to identify the ligation procedure.

5. Add ‘A’ to FA-2X-MS2 with rTaq mix.

6. Ligation of the product of adding ‘A’ and then transform the product into Top 10 competent cells.