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From 2012.igem.org

Week 3 (07.02-07.08): Co-transformation of pCJDD0, pCJDFA, pCJDFB

July 02

1. Prepare LB plates of chloramphenicol resistance.

2. Sequencing of FA-6-29-2 and FA-6-29-2 failed.

July 03

1. Amplify pCJDFA (DH5α) from bacteria of June 29 for sequencing.

2. Co-transformation of pCJDD0, pCJDFA, pCJDFB into BL21*(DE3). Besides, transformation of pCJDD0, pCJDFA, pCJDFB into BL21*(DE3) respectively.

July 04

1. Pick up single colony of plate of D0 (BL21*(DE3)) of July 03 and amplify them in liquid LB medium.

2. Pick up single colony of plate of co-transformation of pCJDD0, pCJDFA, pCJDFB (BL21*(DE3)) of July 03 and amplify them on other LB plate.

July 05

1. Amplify co-transformation bacteria of July 04, FB bacteria of July 03 and E.coli BL21*(DE3) strain in liquid LB medium.

2. Make glycerol stock of D0 (BL21*(DE3)).

3. Transform pCJDFA into (BL21*(DE3) for transformation of July 03 failed.

July 06

1. Measure growth curve of D0 (BL21*(DE3)) and BL21*(DE3), respectively. We mistook the absorbance wavelength of spectrophotometer NanoDrop.

2. Pick up a single colony of plate of FA (BL21*(DE3)) of July 03 and amplify it in liquid LB medium.

3. Pick up a single colony of plate of co-transformation of July 04 and amplify it in liquid LB medium.

4. Amplify co-transformation bacteria of July 06 E.coli BL21*(DE3) strain in liquid LB medium for measurement of growth curve.

5. Make glycerol stock of FA (BL21*(DE3)), FB (BL21*(DE3)) and co-transformation (BL21*(DE3)).

6. Amplify backbone pSB1C3 by amplifying part in plate 4, 1E, 3E. Transform them into DH10β.

July 07

1. Miniprep plasmid of pCJDFB from bacteria of July 06.

Name: FB-7-07 Concentration: 14.6 ng/ul A260/280: 1.73

Name: Co-trans-7-07 Concentration: 9.7 ng/ul A260/280: 1.79

2. Make the first part K738000, RNA scaffold with promoter and terminator. Do PCR procedure.

PCR of D0 seems to be correct, but there is nothing in pCJDFB and the plasmid of co-transformation bacteria is the same as that of pCJDD0.

3. Re-transformation:

No. name resistance plate strain

1 co-transform-2-1 A+K+S Sp BL21*DE3

2 co-transform-2-1 A+K+S Kan BL21*DE3

3 orginal FA Sp Sp DH10β

4 orginal FB Kan Kan DH10β

5 FA-6-30-1 Sp Sp DH10β

6 FB-7-01-4 Kan Kan DH10β

7 plate3 16H Kan Kan BL21*DE3

When coated the plates, we made a mistake an put No.3 and No.4 bacteria together on Sp resistance plate, thus somehow leading to failure of these two transformations.

4. PCR purification of D0 PCR product.

5. Inoculate D0 bacteria in 3 resistance liquid LB medium to see if there is problem with antibiotics.

6. Double digest D0-PCR-product and pSB1C3 with EcoR1 and Pst1.

7. One of our teammate went to Peking and communicate with iGEM team there.

July 08

1. Measure growth curve: inoculate co-transformation 2-1 bacteria 1 ml into 50 ml liquid LB medium. Measurement was done with 723-Spectrum. It grew so slow that we gave up again.

2. Test the efficiency of competent cells with part in plate 1, 1G.

3. Amplify FA-7-3-1 and FA-7-3-2 bacteria for minipreparing plasmids later.

4. Miniprep plasmid of pCJDFB from bacteria of July 06.

Name: FA-7-8-1 Concentration: 54.1 ng/ul A260/280: 1.88

Name: FA-7-8-2 Concentration: 45.1 ng/ul A260/280: 1.89

The concentration is the highest among all pCJDFA we have miniprepared by now. However, its electrophoresis result is not correct at all.

5. Amplify glycerol stock bacteria FA (BL21*(DE3)) and FB (BL21*(DE3)) on plates.

6. Purify the products of double digestion of July 07.