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August 5th - Performing PCR gradient for alpha fragment and omega fragment with and without linker ○ Temperatures used for the PCR are 510C, 54.50C, 60.20C, 63.90C, 670C and 69.50C - Running electrophoresis to check the result ○ The bands were very thin, so we decided to re-PCR the alpha and omega fragment August 6th - Linearizing LacZ full from 3rd coloni in the replica (third transformation) - PCR alpha and omega fragment with and without linker ○ Temperatures used for the PCR are 500C, 53.20C, 55.50C, 61.00C, 66.80C, and 68.40C - Running electrophoresis ○ The thickest band was shown at 53.20C for alpha fragment and at 500C for omega fragment. Hence the temperatures said above will be used for PCR using Taq HiFi August 12th - Running ○ We got bands with correct length, however we got thin bands on well containing omega fragment of beta galactosidase. We decided to do large scale PCR using PFx the next day. August 13th - Large scale PCR (50μL) using PFx enzyme with annealing temperature at 53.20C for alpha fragment, 500C for omega fragment and omega fragment+linker - Running electrophoresis ○ We got the correct length for all fragments August 14th - Large scale PCR (50μL) using PFx enzyme with annealing temperature at 53.20C for alpha fragment, 500C for omega fragment and omega fragment+linker - Running electrophoresis: ○ Correct length for all fragments - Restricting pQE80L using PstI ○ We use three tubes, each of them containing PstI with different volume: 0.5 μL, 5 μL, and without any enzyme added for negative control. § This step was done in order to check the enzyme, because we are using PstI that had past its expiration date. - Running the restricted PCR ○ The result shows that the PstI is no longer in a good condition. For 1 hr incubation period, the restriction using 0.5μL PstI does not produce the correct length of pQE 80L (4751 bp) - Incubating the restricted pQE80L overnight ○ This step was done in order to check the activity of the enzyme in extended period. August 15th - Moving the pQE 80L + PstI into -400C freezer - Purifying the fragments (alpha+linker, omega, omega+llinker)from the PCR products using Low Melting Point Agarose (LMA) method. August 16th - Running the incubated pQE 80L + PstI The enzyme works with extended time of incubation. However, star activity is also observed. Hence, we look up on the internet and found that PstI can still works for extender period of incubation up to 4 hours. August 17th - Checking the isolated fragments' concentration using Nanodrop 2000 ○ Alpha fragment concentration: 17.1 ng/μL ○ Omega fragment concentration: 18.8 ng/μL ○ Omega+linker fragment concentration: 17.1 ng/μL - Ligating pSB1C3 and omega fragment August 18th - Running purified plasmid in agarose - Linearizing alpha fragment using PstI - Ligating pcDNA 3.1 + omega fragment - Ligating pcDNA 3.1 + alpha fragment - Cutting the ligated psB1C3 + omega fragment using EcoRI to verify he length of fragment inserted - Running August 19th - Re-ligating psB1C3+ω - Cut pCDNA+α and pCDNA+ω with BamHI - Running August 20th - Running the ligated parts - Making new competent cells - Transforming the ligated parts - Spread the transformed cells into agar plates August 22nd - Making agar plates - Re-PCR alpha and omega fragments using PFX HiFi - Running electrophoresis August 23rd - Ligating gBlocks to pBluescript and pSB1C3 - -Ligating alpha and omega fragment to pBluescript - Purify PCR products using LMA - Ligating alpha and omega fragments from PCR products to pcDNA 3.1 August 24th - Nanodrop the LMA product - Nanodrop the previously isolated alpha fragment - Restricting LMA with omega fragments using EcoRI - Restricting pCDNA 3.1 using EcoRI and XhoI - Restricting the previously purified alpha fragment using EcoRI - Running August 25th - Restricting: ○ LMA omega fragmetns ○ Lox511 gBlock ○ (G4S)4 gBlock - Purifying the restricted plasmid from the preious day - Ligating ○ Omega fragment-pCDNA 3.1 ○ Alpha fragment-pCDNA 3.1 ○ Omega fragment-pSB1C3 ○ Lox511-pSB1C3 ○ (G4S)4- pSB1C3 - Purifying the following: ○ Omega fragment restricted with EcoRI and PstI ○ Lox511 ○ (G4S)4 August 26th - Transforming ○ Lox511 ○ (G4S)4 ○ pQE 80-L ○ pQE 82-L ○ Positive control for ampicillin and chloramphenicol antibiotics - Spread - Streak E.coli top10 - Ligation using T4 DNA Ligase Promega + T4 NEBuffer August 27th - Making replica on LB agar plate - Running the previously ligated plasmids - Restriction using BamHI and PstI - Purification of DNA - Making new agar plates - Making overnight top10 - Inoculating bacteria in LB Broth for mall scale isolation August 28th - PCR omega fragments - Small scale isolation - Restriction using EcoRI to check the length - Running August 30th - Inoculating bacteria with pBluescript KS in 4 mL LB broth - PCR omega fragment - Making LB Agar - Making LB Broth - Making pBluescript KS replica August 31st - Small scale isolaiton Running PCR productsAugust 30th - Inoculating bacteria with pBluescript KS in 4 mL LB broth - PCR omega fragment - Making LB Agar - Making LB Broth - Making pBluescript KS replica August 31st - Small scale isolaiton - Running PCR products September 1st - Purification of PCR omega fragment - Making gene ruler - Running the purified product - Nanodrop the purified product : 110 ng/μL - Inoculating bacteria with pBluescript KS and pSB1C3 containg alpha fragment - Spreading BL21 and Top10 on agar plates with chloramphenicol antibiotic to check the optimum chloramphenicol concentration. September 3rd - Nanodrop the large scale isolation product: ○ pBluesript KS: 69.5 ng/μL ○ Alpha fragment : 179.5 ng/μL - Restricting pBluescript KS using EcoRv - Nanodrop the restricted plasmid - Making competent cells September 4th - Testing te chloramphenicol concentration - Testing the enzymatic activity from PstI - Making agar plates with different concentration of chloramphenicol - Running the result of enzymatic activity test September 5th - Re-testing the enzymatic activity using different buffers - Running - Checking the chloramphenicol test result - Transforming: ○ Omeg fragment-pBluescript KS ○ (G4S)4 -pSB1C3 (ligated 3/9) ○ Lox511-psB1C3 (ligated 3/9) ○ Lox511-psB1C3 (ligated 25/8) ○ Lox511-psB1C3 (ligated 25/8) ○ (G4S)4 -pSB1C3 (ligated 25/8) ○ pQE80L ○ Re-ligating omega fragment-pSB1C3 September 6th - Restricting: ○ Omega fragment + EcoRI ○ pSB1C3 ○ Omega fragmetn +XbaI ○ pBluscript KS + XbaI ○ Alpha fragment + XbaI - Running - Nanodrop the purified digested plasmid ○ Omega fragment + EcoRI = 7.2 ng/μL ○ pSB1C3 = 3.8 ng/μL ○ Omega fragment + XbaI = 7.7 ng/μL ○ pBluescript KS + XbaI = 9.8 ng/μL ○ Alpha fragment + Xba I = 0.35 ng/μL - Running - Inoculating bacteria with pSB1C3-alpha fragment in 100mL broth September 9th - Large scale pSB1C3-alpha fragment isolation - Restricting pBluescript KS using EcoRV - Digesting linearized pSB1C3 and linearized omega fragment using PstI - Making 750mL LB Broth - Making agar plates - Digesting pSB1C3-alpha fragment - Purifying the digested using LMA - Blunting Lox511 and (G4S)4 linker - Running September 11th - Small scale isolation - Running the small scale isolation product September 13th - Re-run the small scale iolation product - Inoculate the bacteria containing pSB1C3=omega fragment September 14th - Running small scale isolation product - PCR Colony - Large scale isolation - Re-ligating omega fragment with pSB1C3 September 15th - Running ○ Digested pQE+PstI ○ PQE ○ Digested alpha fragment+PstI ○ Alpha fragment ○ Marker ○ 5 ○ 8 ○ 9 ○ 15 ○ Large scale isolation pBluescript KS-omega

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