The first step of the experiment is to add the design and then add the restriction enzyme (RE), in this case EcoRi, to the insert in order to recognize the target sequence within double stranded DNA and cut both strands of the DNA. After this, the single stranded nucleotide tails of the both EZH2 and pBluescript can be combined together.
A representation of how the restriction enzyme recognize and cuts the site
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(1) https://www.khanacademy.org/science/biology/biotech-dna-technology/dna-cloning-tutorial/a/restriction-enzymes-dna-ligase
In order to design EcoRI, the plasmid vector has to be digested and the PCR has to be inserted into the DNA.
Table 1: The steps for the digestion of the vector and insertion of PCR into DNA
Cycle Description
Plasmid vector digest Digest 2ug of the plasmid with 1ul of EcoRI and the rest of the components from Figure 2; then use 1ul of enzyme and digest the new product, pBS.E (pBS enzyme/linearized pBS) for 60 mins at 37°C
Setting up PCR on EZH2 (?)
Inactivation of RE Heat the enzyme to 65°C for 20 mins to
A representation of the EZH2 and the amino acids produced
Table 2: The volumes of the components that make the restriction enzyme
Component Volume (uL)
DNA 8
RE buffer 2.5
dH2O 13.5
RE 1
Total 25
After the components have been mixed, the next part is to incubate the ………………………………………..
EZH2 gene-0.05 pg/ul
PCR mix:
Table 3: Volumes of the components used to make the PCR mix
Component Volume (uL)
pGEM.EZH2 30
Forward primer (10uM) 3
Reverse primer (10uM) 3
Enzyme buffer+taq 40
dH2O 4
Total 80
Vector dephosphorylation:
The volume of pBS.E was increased to 40ul by adding water and then 28ul of this was removed into a new microfuge tube labelled pBS.ES (linearized, dephosphorylated/SAP treated pBS). Then it was incubated at 37°C for 60 mins and later, the enzyme was inactivated by heating it for 15 mins at 75°C. At the end, a volume of 12ul of pBS.E (RE digested plasmid) was saved.
Digesting the PCR product:
Table 4: EZH2 PCR cycling conditions and components
Cycles Time Temperature (°C) Process
Cycle 1 3 mins 95
35 cycles 1 min 95 Denature
1min 60 Anneal primers
1min 72 Extend with Taq
Polymerase
Final cycle 10 mins 72 Final extension with Taq
Purifying the PCR product:
For this part of the experiment a QIAquick column was used to purify the PCR.
After calculations, a volume of 67.8 ul of PCR was added to 339ul of Buffer PB and mix. A QIAquick spin column was then placed in a 2 ml collection tube, then the previous mixed was added and centrifuged for 1 min. After discarding the flow through, this step was repeated one more time and another 0.75 ml Buffer PE was added to the solution and centrifuged for 1 min. After discarding it again, at the final step, 40ul elution buffer was added and centrifuged again and the new tube was labelled PCR.E (PCR DNA digested with RE).
At this moment there should be 4 samples: 3 from the plasmid (pBS, pBS.E and pBS.ES) and the PCR.E.
Sizing and quantifying DNAs using agarose gel:
After the agarose gel was set up, the following samples of DNA were loaded into the gel, in order:
Table 5: The volumes of the samples added onto the agarose gel
Sample Specific component Water Loading dye
Digested PCR product PCR- 5ul 5ul 3ul
NEB 2-log ladder Mw marker Pre-mixed DNA ladder/dye- 10ul
pBS.ES DNA sample-5ul 5ul 3ul
pBS Undigested plasmid (pBS)- 1ul 9ul 3ul
After the DNA samples were loaded, electrophoresis started for 5 mins at 50V, followed by the increasing the voltage power to 100V for 60 mins. After this step was finished, an image of the gel was recorded.
DNA ligations and bacterial transformations:
A main component for the fusion of those 2 is T4 DNA ligase, an enzyme that fuses the ends of the stranded overhands together into 1 single and unbroken molecule of DNA.
A diagram that shows how DNA ligase works – (1)
At this step, 3 ligation reactions were performed, each with a different molar ration plasmid and insert.
Table 6: Volumes of the components for each of the 3 ligation reactions
Reaction Ligase Buffer pBS version T4 DNA Ligase PCR volume (ul) PCR insert
ratio H2O volume (ul)
1 7.5 pBS-0.2 0 0 – 5.3
2 7.5 pBS.E-1 0 0 – 6.5
3 7.5 pBS.E-1 1 0 – 5.5
4 7.5 pBS.ES-1.4 1 0 – 5.1
5 7.5 – 0 5 – 2.5
6 7.5 pBS.ES-1 1 1 1:3 0.5
7 7.5 pBS.ES-1.4 1 1 1:1 3.2
8 7.5 pBS.ES-2.8 1 1 3:1 2.5
9 7.5 pBS.ES-1.4 1 1 – 4.1
ADD SOME INFO ABOUT THE LIGATION MAYBE!!!!!!!!!!!!!!!
The next step is transformation where the plasmid was inserted into E.coli by heat-shocking the bacterial cells in order to improve the migration.
For this,the ligation reactions were incubated at room temperature to improved the mix of the DNA molecules into the bacterial cells. They were then placed on the ice and a volume of 100 ul of competent cells was added to each tube and mixed. After they were incubated for 15 mins, they went through a heat-shock stage for 2 mins at 45°C. The last step was adding 0.9 ml of L-broth to each tube and incubating them for 45 mins at 37 °C, followed by labelling them.
The next part of this step was finding the bacterial cells that contain the plasmid and are antibiotic-resistant by counting the white and blue colonies from each plate. A small amount of the colonies from each plate 6-9 was added to a 2ml liquid broth and let to grow overnight at 37 °C.
Plasmid purification and restriction digest:
………………………………………..
Table 7: The volumes (in ul) of components containing and missing HindIII
Component Recombinant plasmid with HindIII Recombinant plasmid without HindIII pBS with HindIII pBS without HindIII
DNA 5 5 2 2
RE Buffer 1 1 1 1
dH2O 3 4 6 7
RE 1 0 1 0
Total 10 10 10 10
Gel to identify insert:
The last stage of the gene cloning experiment was running the electrophoresis with the samples in order to determine the size of the DNA and to visualize the gel on a UV-transilluminator.
