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 products 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 products

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