Difference between revisions of "Team:TCU Taiwan/Notebook/Lab"

June 7th ,2015

• Cultured cells   < Exchange medium >     1. Suction whole medium     2. Add3-5c.c.PBS, and shaking gently to clean     3. Suck out PBS     4. Add 7-10c.c. medium     5. Returned to the incubator

June 13rd ,2015;

• The senior teach us to Bacterial culture.

June 14th ,2015

• The senior teach us plasmid purification.

June 15th ,2015

• The senior teach us to test the concentration of the plasmid.

July 1st ,2015

• We seeded E.coli DH5α which have plasmid p-BluescriptⅡSK(-).

July 2nd ,2015

• Isolated of p-BluescriptⅡSK(-) plasmid for twenty tubes and detected the plasmid concentration to make sure whether we isolated the plasmid . •Add restriction enzyme speⅠ to the one of twenty plasmid tubes , the tube #4 , in order to produce vector , and run gel electrophoresis for detection . • The senior brother teach us how to do restriction enzyme cutting • Due to produce more plasmid,we decide to do again bacterial culture • p-Biuescript plasmid purification by ourselves

p-Biuescript plasmid digest with Spe I

culture DH5α with p-Biuescript plasmid

July 3rd ,2015

• Pumping plasmid.But the result is not ideal , it was decide to do again bacterial culture next day.

July 4th ,2015

• Professor Ji Hshiung Chen teach us to do restriction enzyme cutting effectively. • Bacterial culture • Culture DH5α with p-Biuescript plasmid

July 5th ,2015

• Pumping plasmid • Bacterial culture

p-Biuescript plasmid purification

p-Biuescript plasmid digest with Spe I

July 6th ,2015

• Pumping plasmid • Professor Ji Hshiung Chen teach us the methods to pump the plasmid sooner.

July 7th ,2015

• We make the speⅠ and the EcoRⅠ to cut plasmid. • According to the method by Professor Ji Hshiung Chen,try theenzyme Spe I and the EcoR I from differnt brands.

July 8th ,2015

• We make the speⅠ and the EcoRⅠ to cut plasmid.

July 12nd ,2015

• The senior teach us transformation，pQE60 transform into DH5α

July 13rd ,2015

• Culture DH5α with pQE60

July 14th ,2015

• Pumping the plasmid of PQE60 • Bacterial culture

pQE60 plasmid purification

culture DH5α with pQE60

July 15th ,2015

• pQE60 plasmid purification

July 17th ,2015

• We make the enzyme BamHⅠ and the enzyme NcoⅠ to cut plasmid. • pQE60 plasmid digest with enzyme BamH I and Nco I

July 18th ,2015

• We make the enzyme BamHⅠ and the enzyme NcoⅠ to cut plasmid. • pQE60 plasmid digest with enzyme BamH I and Nco I • Gel extraction pQE60 with BamH I and Nco I digesting

July 20th ,2015

• Restriction enzyme cutting • pQE60 plasmid digest with enzyme BamH I and Nco I

July 21st ,2015

• pQE60 plasmid digest with enzyme BamH I and Nco I

July 22nd ,2015

• gel electrophoresi

July 23rd ,2015

• Gel extraction pQE60 with BamH I and Nco I digesting

July 25th ,2015

• Gel extraction pQE60 with BamH I and Nco I digesting

July 27th ,2015

• Practice for the first animal model

July 28th ,2015

• Practice for the second animal model

July 31st ,2015

• Help the mice to change gressing for the first practice

Aug. 1st ,2015

• Help the mice to change gressing for the second practice

Aug. 5th ,2015

•  * PCR signiferin

2% gel. The PCR product of signiferin should be 100~200 bp. But, there isn’t any band on it. Therefore, we consider that we PCR fail.

Aug. 6th ,2015

•  * PCR signiferin

2% gel. The PCR product of signiferin should be 100~200 bp. As the result, there is band between 100~200 bp on sample 2 so we consider it is correct.

Aug. 7th ,2015

•  * PCR signiferin

Aug. 9th ,2015

•  * PCR signiferin

Aug. 11st ,2015

•  Gel extraction signiferin

Aug. 12nd ,2015

•  ligation signiferin into TA clone

Aug. 13rd ,2015

•  transformation (TA clone with signiferin)

Aug. 14th ,2015

•  Culture DH5α with pQE60

Aug. 15th ,2015

•  signiferin TA plasmid purification

0.8% gel. The size of TA plasmid with signiferin insert should be 2000~3000 bp. In addition, plasmid will become sorpercoil so it will run faster. Therefore, we consider these are the correct plasmid.

Aug. 16th ,2015

•  pQE60 plasmid digest with enzyme BamH I and Nco I biobrick digest with enzyme EcoRI and Pst I

2% gel. The size of signiferin insert should be 100~20 bp. In addition, the signiferin insert in biobrick should be a little bigger than in pQE60. As the result, these two are all the correct sample we need.

Aug. 17th ,2015

•  gel extraction of signiferin insert

Aug. 18th ,2015

•  gel extraction of pQE60 vector • Transform Epi-1 plasmid into DH5α • Ligate pQE60 vector and signiferin insert

Aug. 19th ,2015

•  backbone enzyme digestion •  Ligate backbone vector and signiferin insert •  Culture DH5α with Epi-1 plasmid • Transform pQE60 vector with signiferin insert into DH5α

Aug. 20th ,2015

•  plasmid purification of pQE60 vector with signiferin insert

0.8% gel. There are band on sample 1~7 of pQE60 plasmid with signiferin insert, but the size of our plasmid should be 3000 to 4000. As the result, these samples are not the plasmid we need.

Aug. 21st ,2015

•  PCR Epi-1 insert

2% gel. It is the correct size that Epi-1 PCR product have band between 200~300 bp. But, there is also band on blank. We consider it was polluted when we add reagent.

•  ligate TA vector and Epi-1 insert •  plasmid purification of pQE60 vector with signiferin insert

0.8% gel. The size of our plasmid should be 3000 to 4000. As the result, these samples are the plasmid we need.

•  enzyme digestion to check the clone of pQE60 vector with signiferin insert

2% gel. The signiferin insert should have band between 100~200 bp. We consider that maybe we didn’t ligate it into pQE60.

Aug. 22rd ,2015

•  transform TA vector with Epi-1 insert into DH5α •  Transform pQE60 vector with signiferin insert into DH5α •  Plasmid purification of pQE60 plasmid with signiferin insert •  Plasmid purification of backbone with signiferin insert

0.8% gel. The size of signiferin biobrick should be 2000~3000 bp, and the size of pQE60 plasmid with signiferin should be 3000~4000 bp. In addition, the plasmid will become surpercoil so it will run faster. Therefore, they are all correct plasmid.

Aug. 23rd ,2015

•  enzyme digestion to check the clone of pQE60 vector with signiferin insert

2% gel. The size of signiferin insert should be 100 to 200 bp. But, there are no band on each sample. We consider that the signiferin insert have not been ligated.

•  Enzyme digestion to check the clone of biobrick with signiferin

2% gel. The size of signiferin insert should be 100 to 200 bp. There is band on the sample 2 of enzyme digest product. As the result, we can make sure that the signiferin has been ligated in backbone.

•  Culture TA clone with Epi-1 insert • Culture pQE60 with signiferin insert • Backbone digestion

Aug. 24th ,2015

•  Plasmid purification of pQE60 plasmid with signiferin insert

0.8% gel. The size of pQE60 plasmid with signiferin should be 3000~4000 bp, and the plasmid will become surpercoil so it will run faster. Therefore, there was no correct sample. Plasmid purification of Epi-1 TA clone Plasmid purification of signiferin biobrick

Aug. 25th ,2015

•  Enzyme digestion (TA clone of Epi-1)

2% gel. The size of Epi-1 is about 200 to 300 bp. There are bands on sample 2 and 3. As the result, it have ligated into TA clone. Gel extraction Enzyme digestion to check the clone of biobrick with signiferin Bacteria culture (signiferin biobrick, signiferin pQE60, TA clone of signiferin, TA clone of Epi-1)