PCR
Polymerase chain reaction (PCR) is a laboratory technique for rapidly producing (amplifying) millions to
billions of copies of a specific segment of DNA, which can then be studied in greater detail. PCR involves
using short synthetic DNA fragments called primers to select a segment of the genome to be amplified, and then
multiple rounds of DNA synthesis to amplify that segment.
Principle to replicate a targeted sequence:
DNA targeted sequence denatures to two single strands at 98 degrees.
Temperature lowers, top and bottom primers are added to anneal the templates.
A type of enzyme helps join free bases to make elongate the double stranded DNA.
Steps are repeated to give 2n strands of DNA, with n being the number of replications.
Components added:
Mix buffer-
dyed nucleotides(atcg)-5microlitres
Primers-
Primer 1 and primer 2-2microlitres*2
DNA-
targeted sequence(acc(1,2),fast 1,fast 2)-1 microlitre
Distilled water-
ddH2O-10 microlitres
Method:
- Primer is put into the centrifuge (get liquid off the sides);
- Use specific micropipettes to add the components into PCR tube;
- Complete solution is vibrated to mix evenly;
- Complete solution is put into the centrifuge;
- Complete solution is put into the PCR machine at a set temperature cycle (Repeated for the three different
enzymes).
Analyze PCR
Make Electrophoresis:Nucleic acid dye, water, and TAE is microwaved to make an agarose gel
electrophoresis.
Electrophoresis:Run for 30 mins
See the results:using the 8k DNA marker, read the results shown
Gel Purification
To use Gel DNA Extraction Kit to extract ACC1, FAS1, and FAS2.
- Cut blocks containing the target fragment from the agarose gel into centrifuge tubes.
- Weigh the amount needed.
- Add 3-6 times the weight of Gel to Buffer B2, 50 degrees water bath for 5-10 minutes to dissolve the
gel.
- For fragments smaller than 500 bp, add one-third of the volume of buffer b2 with isopropanol.
- Transfer the lysate into the adsorption column and centrifuge at 8000xg for 30 seconds. Pour off the
liquid in the collection tube.
- Add 500 µL wash solution, centrifuge at 9000xg for 30 seconds, pour off the liquid in the collection
tube.
- Empty the adsorption column centrifuged at 9000xg for 1 minute.
- Place the adsorption column into a clean 1.5 mL centrifuge tube, add 15-40 µL of Elution buffer to the
center of the adsorption membrane, let it stand at room temperature for 1 minute, then centrifuge for one
minute to save the DNA solution in the tube.
DNA Recombination
- Prepare: 2 uL ddH20, 5uL ClonExpress Mix, 2 uL linearized vector, 1uL insert.
- Mix gently by pipetting (do not shake to mix) and collect the reaction solution to the bottom of the tube
by
brief centrifugation.
- Single fragment reconstitution reaction, 50°C, 5 min: reduce to 4C or cool immediately on ice.
Transform the target genes into Escherichia coli:
- Place chemo-sensitive cells for cloning on ice to thaw.
- Add 5-10 µL of the recombinant product to 100 µL of the chemoreceptor cells, flick the wall of the tube to
mix, and let stand on ice for 30 minutes.
- Heat stimulate in a 42℃ water bath for 45 seconds, then immediately cool on ice for 2-3 minutes.
- Add 900 µL of SOC or LB liquid medium and shake at 37℃ for 1 hour.
- Pre-warm the corresponding resistant LB solid medium plates in a 37℃ incubator.
- Centrifuge at 5000 rpm (2500xg) for five minutes and discard 900 µL of supernatant. Use the remaining
medium to re-suspend the bacteria and apply gently with a sterile applicator stick on the plate containing
the correct resistance
- Incubate upside down in a 37℃ incubator for 12-16 hours
Detecting monoclonies
Pick monoclonal (receptor cells)
Use the micropipette tips to take out a colony and add it to the LB medium.
Oscillate it for 12-15 hours to copy and duplicate itself.
- Add mix, ddH2O, two primers, and the colony of ACC1, FAS1, FAS2 into a PCR tube
- Put into PCR machine to amplify and copy.
Making PCR gels:
Agarose gel electrophoresis
- TAE (powder melted in 1L ddH2O).
- 0.24 g agarose (0.8% agarose gel) + 30 mL 1x TAE.
- Microwave oven on high for one minute.
- Add a small amount of nucleic acid fuel.
- Pour into mold and solidify.
- Weigh 135 volt electrophoresis for 30 minutes.
Run the Gels:
Add the marker, FAS1, FAS2 into the electrophoresis.
Run the electricity and wait for 30mins.
Check results
There were no results due to a error from one of the steps ahead.