Team:TCU Taiwan/Notebook/Lab

Lab

June 29th 2016 *Practice:

         1.Preparing competent cell

July 4th 2016 *Practice:

         1.Transforming PQE60 plasmid into the colony of competent cell

June 15th 2016 *Practice:

         1.Culturing the competent cell by using streak plate methods

June 17th 2016  *Practice:

         1.Culturing the colony of the competent cell from streak plate methods by picking a colony and put it into the LB

June 18th 2016 *Practice:

         1.Plasmid extraction from the cultured colony which contains PQE60 plasmid

         2.Run the gel for checking the presence of PQE60

August 7th 2016:

         1.Culturing E. coli BL21 competent cell by using streak plate methods and spread plate methods

August 10th 2016:

         1.Transforming plasmid PQE60 (red chromo protein) into the E. coli BL21 competent cell

         2.Preparing LB agar media for culturing the bacteria

         3.Culturing PQE60 inside of the E. coli BL21 competent cell with spread plate methods using same concentration of selective marker (ampicillin)

August 17th 2016:

         1.PQE60 Plasmid Extraction

         2.Run Gel Electrophoresis (Result: Failed)

         3.Culturing the PQE60 inside the competent cell using spread plate methods

August 20th 2016:

         1.Plasmid Extraction

         2.Gel electrophoresis (Result : Failed)

August 21st 2016:

         1.Transformation red color chromo protein into competent cell

         2.Culturing PQE60 in the E. coli BL21 competent cell using different concentration of selective marker

August 28th 2016  *Practice:

         1.Bacterial genomic DNA isolation

September 1st 2016 :

         1.Making a lot of DNA isolation from the E. coli BL21 which is contain ptsG sequence

         2.Performing of PCR Hxt1 sequence from yeast

         3.Checking Bacterial genomic DNA concentration with Nano drop

         4.Performing of PCR ptsG sequence from the Isolated DNA of E.coli

         5.Gel electrophoresis (Result : failed)

         6.Picking a colony of different selective marker and culture it in the LB media for over night

         7.Practice:PCR

September 3rd 2016:

         1.Plasmid Extraction of different concentration of selective marker

         2.Gel Electrophoresis (Result : Failed, band located at the unwanted place)

September 8th 2016:

         1.Transformation PQE60 into the E. coli BL21

         2.Transformation pSB1C3 into the E. coli BL21

         3.Culturing transformed PQE60 and pSB1C3 with spread plate methods with different concentration of selective marker

September 14th 2016:

         1.Picking up a colony of PQE60 and pSB1C3

         2.Culturing Colony in the LB media for over night

September 15th 2016:

         1.Plasmid Extraction from the cultured colony of PQE60 and pSB1C3

         2.Bacterial genomic DNA isolation from E.coli

         3.PCR for ptsG sequence

         4.PCR purification of ptsG sequence

         5.Gel electrophoresis (result: Succeed)

         6.Performing restriction enzyme digestion for ptsG sequence

         7.Practice:Using PCR purification KIT

September 16th 2016:

         1.Performing PCR for ptsG sequence

         2.Run the Gel electrophoresis in a lot of ptsG sequence amount

         3.Practice:Using Gel purification KIT

September 17th 2016:

         1.Gel Purification of where the ptsG band exist

September 19th 2016:

         1.Measuring the O.D. value of cultured PQE60 ( red color chromo protein)

September 20th 2016:

         1.Doubling the sample of the ptsG sequence by using PCR

September 21st 2016:

         1.Plasmid Extraction of PQE60 plasmid

         2.Cutting PQE60 using restriction enzyme BamHI

         3.Cutting PQE60 using restriction enzyme NCoI

September 22nd 2016 :

         1.Realize forward primer and reverse primer for ptsG is wrong.

         2.Redesign the forward and reverse primer

         3.Genomic DNA isolation from E.coli

September 27th 2016:

         1.Cutting pSB1C3 plasmid with restriction enzyme XbaI and SpeI

September 29th 2016:

         1.PCR for ptsG sequence

         2.PCR for red color chromo protein

September 30th 2016:

         1.Cutting PQE60 (red color chromo protein with BamHI and NcoI)

         2.Cutting pSB1C3 (red color chromo protein with EcoRI and pStI)

         3.PCR of Cut pSB1C3 (red color chromo protein)

October 1st 2016:

         1.Cutting PQE60 with NcoI and HindIII

October 2nd 2016:

         1.Ligation PQE60 (cut by NcoI and BamHI) with ptsG sequence

         2.Ligation PQE60 (cut by NcoI and BamHIII) with ptsG + red color chromo protein sequence

         3.Transformation the ligation product (ptsG in the PQE60 ) into BL21

         4.Transformation the ligation product (ptsG + red color chromo protein) into E. coli BL21

October 3rd 2016:

         1.Ligation of PQE60 (cut by NcoI and BamHIII) with ptsG + red color chromo protein

         2.Ligation of PQE60 (cut by NcoI and BamHIII) with ptsG for negative control

October 4th 2016:

         1.ptsG + red color chromo protein PCR

October 5th 2016:

         1.Cutting PQE60 with NcoI and HindIII

         2.Cutting pSB1C3 with EcoRI and pstI

October 7th 2016:

         1.PCR for ptsG inserted in the pSB1C3

         2.PCR for red color choromo protein inserted in the pSB1C3

         3.PCR purification for ptsG and red color chromo protein after PCR

         4.Run the gel of both ptsG and red color chromo protein

         5.Gel purification for both ptsG and red color chromo protein

October 8th 2016:

         1.Ligation of pSB1C3 (cut by EcoRI and pstI) with ptsG sequence

         2.Ligation of the ptsG and red color chromo protein inside the PQE60 (cut by NcoI and HindIII)

         3.Cutting pSB1C3 by EcoRI

         4.Ligation ptsG (for pSB1C3) and red color chromo protein (for pSB1C3) with pSB1C3 plasmid

         5.PCR ptsG + red color chromo protein for pSB1C3

         6.Ligation ptsG (for PQE60) and red color chromo protein (for PQE60) with PQE60 plasmid

October 9th 2016:

         1.Cutting PQE60 with BamHI for inserted in PQE60

         2.Cutting red color chromo protein with BglI and HindIII for inserted in PQE60

         3.Cutting PQE60 with BglI and HindIII

October 10th 2016:

         1.Checking the ability urine as the media for E.coli to grow by using 3 kinds of media: LB, LB + urine, Urine.

         2.Measure O.D. value of the E.coli concentration for different culturing media.

October 11th 2016:

         1.PCR of the fusion protein of the ptsG + red color chromo protein for PQE60

         2.Ligation ptsG + red color chromo protein with pSB1C3

         3.Ligation ptsG + red color chromo protein with PQE60

         4.Transformation of the pSB1C3 which is already contain ptsG + red color chromo protein

         5.Transformation of the pSB1C3 to be send for iGEM

October 12th 2016:

         1.PCR of the fusion protein (ptsG + red color chromo protein)

         2.using NcoI to cut PQE60

         3.using HindIII to cut PQE60

         4.using EcoRI to cut pSB1C3

         5.Ligation fusion protein with PQE60

         6.Cutting fusion protein to be ligated with PQE60

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