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:

  1. Primer is put into the centrifuge (get liquid off the sides);
  2. Use specific micropipettes to add the components into PCR tube;
  3. Complete solution is vibrated to mix evenly;
  4. Complete solution is put into the centrifuge;
  5. 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.

  1. Cut blocks containing the target fragment from the agarose gel into centrifuge tubes.
  2. Weigh the amount needed.
  3. Add 3-6 times the weight of Gel to Buffer B2, 50 degrees water bath for 5-10 minutes to dissolve the gel.
  4. For fragments smaller than 500 bp, add one-third of the volume of buffer b2 with isopropanol.
  5. Transfer the lysate into the adsorption column and centrifuge at 8000xg for 30 seconds. Pour off the liquid in the collection tube.
  6. Add 500 µL wash solution, centrifuge at 9000xg for 30 seconds, pour off the liquid in the collection tube.
  7. Empty the adsorption column centrifuged at 9000xg for 1 minute.
  8. 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

  1. Prepare: 2 uL ddH20, 5uL ClonExpress Mix, 2 uL linearized vector, 1uL insert.
  2. Mix gently by pipetting (do not shake to mix) and collect the reaction solution to the bottom of the tube by brief centrifugation.
  3. Single fragment reconstitution reaction, 50°C, 5 min: reduce to 4C or cool immediately on ice.

Transform the target genes into Escherichia coli:

  1. Place chemo-sensitive cells for cloning on ice to thaw.
  2. 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.
  3. Heat stimulate in a 42℃ water bath for 45 seconds, then immediately cool on ice for 2-3 minutes.
  4. Add 900 µL of SOC or LB liquid medium and shake at 37℃ for 1 hour.
  5. Pre-warm the corresponding resistant LB solid medium plates in a 37℃ incubator.
  6. 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
  7. 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.

  1. Add mix, ddH2O, two primers, and the colony of ACC1, FAS1, FAS2 into a PCR tube
  2. Put into PCR machine to amplify and copy.

Making PCR gels:

Agarose gel electrophoresis

  1. TAE (powder melted in 1L ddH2O).
  2. 0.24 g agarose (0.8% agarose gel) + 30 mL 1x TAE.
  3. Microwave oven on high for one minute.
  4. Add a small amount of nucleic acid fuel.
  5. Pour into mold and solidify.
  6. 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.