Team:NWPU/Notes

Laboratory Notes

July 11 2016

The first PCR: scaffoldin,bfd, talb and three vector fragments of a total of six fragments of PCR experiments failed due to the direction error by electrophoresis.

July 12 2016

The second PCR: scaffoldin, bfd, talb and vector fragments of a total of six fragments of PCR, ran electrophoresis normally. Recycle the Gel. Used Gibson connection fragment to link the plasmid, transfered the connection plasmid into Bl21(competent state cell), activated and plated.

July 132016

It was found that the bacteria did not grow on the 12th day. Used Gibson connection fragment to link the plasmid the second time. The plasmid was transferred into the bl21, activated and plated. PCR the third time and modified the PCR annealing temperature from 58 to 55. After electrophoresis, the second fragment of the PCR wasn’t discovered.The fourth time,scaffoldin, BFD, TalB and three vector fragments of a total of six fragments of PCR started again.

July 142016

The bacteria coated on the plate still did not grow on the 13th day. Explored the reasons and found that after the PCR we did not add an enzyme and directly applied Gel Extraction purification. Resulting in DNA fragments have problems, and could not produce resistance, which resulted in that the bacteria did not grow. Let the fouth PCR generated fragments do electrophoresis. The PCR of the second fragment was still not found. We suspect that the temperature affected the results of PCR. For the fifth PCR, PCR was performed only on the second fragment, and the annealing temperature was set to the gradient temperature.

July 152016

The second fragment of PCR was successfully extracted and the correct Gel Extraction purification was carried out. The third time Gibson connection fragment was used as the plasmid.The ligated plasmid was transferred into bl21 competent state and activated plate.The sixth PCR, Scaffoldin, BFD, TalB and vector fragments of a total of six fragments PCR.

July 162016

The bacteria board is still not growing on the 15th day. We re-started the fourth Gibson connection at the same time one-step connection. The ligated plasmids were transferred into the bl21 competent state and the plates were activated after activation. Yesterday's PCR fragments were recycling.

July 172016

The bacteria board is no growth on the 16th day. After checking ,when it is activated after transformation of cells, the use of lb added resistance, affecting the experimental results.Re-connect the fifth Gibson connection and the second one-step connection.

July 182016

Found 17’s the bacteria successfully growed. 4 hours after the bacteria colonies PCR validation and found that the band does not. Re-connect.

July 192016

The bacteria of the 18th day successfully grow. After Colony PCR, the results were normal, keeping bacteria, and plasmid extraction. In the afternoon, it is sended to sequence.

July 202016

Sequencing results back, the preservation of the bacteria coated plate. Therefore, we got three strains with scaffoldin, bfd, talb (hereinafter referred to as S, B, T)

July 212016

S, B, T strains were coated plate culture respectively.

July 222016

On the 21th of the coated plate culture results were checked, S, B, T colonies normal. And then expanded to induce protein expression.

July 232016

The first extraction of S, B, T three proteins.

July 242016

The S, B and T proteins produced on 20th day were concentrated, and the transformed S, B and T bacteria were cultured again.

July 252016

Found the bacteria of yesterday died 3 bottles. The transformed S, B and T strains were cultured and induced protein expression.

July 262016

Extract S, B, T proteins.

July 272016

The concentration of protein extracted from S, B and T was measured and found that the concentration of T protein was not well as expected.

July 282016

The transformed S, B, T bacteria cultured and induced S, B, T protein expression.

July 292016

Extract S, B, T proteins.

July 302016

The concentration of S, B, T protein extracted was measured and the first in vitro reaction was carried out.

July 312016

A vial of liquid phase was prepared. S, B and T were transferred to the culture medium.

August 12016

The protein of S, B and T was extracted, and the recombinant plasmid was constructed and PCR was carried out by using BFD-TALB-TALB large plasmid (BTT). Electrophoresis failed leaded to the target band was not found. S, B, T bacteria transformed and S, B, T protein induced culture.

August 22016

S, B, T protein concentrations were measured. Run the first liquid phase and run the second reaction. BTT large plasmids were constructed for the second PCR.

August 32016

Electrophoresis was performed on the bands of PCR of BTT, and the bands were displayed correctly. The Gibson linker was used as a plasmid, and the ligated plasmid was introduced into the bl21 competent state, and the plate was activated after activation.

August 42016

The bacteria plated the day before did not grow.
        The S, B, T strains were cultured and induced S, B, T protein expression.

August 52016

The bacteria was dead, could not extract protein. The Gibson connection was used to link BTT fragment as plasmid, and the ligated plasmid was transferred into the bl21 competent state, and the plate was activated. S, B and T strains were transformed again and the expression of S, B and T protein was induced.

August 62016

Extract S, B, T protein. The bacteria on the plate did not grow. The resistance of the plate was found to be problematic after exploring the reasons .After the plates were changed , do all things again.

August 72016

The concentration of S, B, T protein extracted from the previous day was measured. The plate coated the day before grew BTT colonies, colonies PCR, plasmid extraction, and sent to the sequencing. A third in vitro reaction was performed and the first in vivo reaction was performed.
       BTT and scaffold double plasmid (hereinafter referred to as sbtt) transformation experiments were performed and coated.

August 82016

The bacteria on the plate did not grow. Sbtt transformation experiments were performed again and plated. Transformed S, B, T again and induced S, B, T protein expression.

August 92016

The bacteria on the plate the day before did not grow, concluded that the competent cell bl21 has problem so we changed it and coated plate culture again.
Extract S, B, T protein.

August 102016

The bacteria on the plate the day before did not grow, assumed that the success rates of simultaneous conversion of the two plasmids is too low. Btt sequencing results came out. Concentrate S, B, T protein and measured the concentration.

August 112016

Carried out vitro experiment again, and electrotransformed two plasmids for twice, coated Sbtt plate again. Expansion of S, B, T, BTT strains.

August 122016

Carried out the vivo experiment for the second time. Sbtt colonies grew successfully.

August 132016

Carried out the btt and sbtt vivo experiment, S, B and T proteins were induced and the expression of S, B and T protein was induced.

August 142016

Extract S, B, T protein.

August 152016

Carried out the btt & sbtt vivo experiment again, measured the concentration of S,B,T

August 162016

Continued the btt & sbtt vivo experiment, S, B and T proteins were induced and the expression of S, B and T protein was induced.

August 172016

Extract S, B, T protein, denied the result of the vivo experiment but still kept on the vivo experiment and developed it further more. We developed the vitro experiment process and carried out overexpression of in vitro reaction product.

August 182016

Liquid phase experiment.
        Condensed the S,B,T protein extracted on the previous day.
        Keep dealing with the vivo experiment and Liquid phase analysis.

August 192016

Took back the vivo reaction bottle, and carried out the final liquid phase test.

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