Gene Synthesis from Oligonucleotides

Templateless PCR

Our goal in this session is to assemble the GFP gene from small pieces of DNA ~60 nucleotides long (called oligonucleotides or oligos). We use templateless PCR (also called Polymerase Cycling Assembly or Assembly PCR) to assemble the oligonucleotides into the full-length GFP gene. While this method effectively assembles the gene, it produces a relatively low yield of the full-length gene and many smaller fragments will also be produced. The Wikipedia site offers a useful introduction to polymerase cycling assembly: [1]

• Before you set up reactions, make sure you reserve a spot in the PCR machine.
• Set up your reactions on ice. Keep nucleotides and master mix on ice AT ALL TIMES!
• Be sure that each reagent is well mixed by pipetting up and down several times before using
  

You will be performing 3 reactions:

1. Assembly of the GFP gene
2. A positive control reaction (a reaction that we know should produce large amounts of DNA)
3. A negative control reaction (a reaction that contains no input DNA and therefore should not produce any DNA; this is a check for contaminating DNA)

Procedure:

1. Obtain 3 PCR tubes (these are the small thin-walled tubes). Label the tubes 1-3.
2. To each tube, add the components listed below.

	Assembly reaction	Positive control reaction	Negative control reaction
PCR master mix (buffer and enzyme)	10 ul	10 ul	10 ul
Nucleotides (1.25 mM)	5 ul	5 ul	5 ul
Oligos	5 ul of assembly oligos	5 ul of control oligos	5 ul of control oligos
DNA template	5 ul of water	5 ul of control template	5 ul of water
Total	25 ul	25 ul	25 ul

3. Cap your tubes and make sure that they are sealed tightly so that the liquid will not evaporate.
4. Place your tubes in the PCR machine in the position for which you signed up. Make sure that you have recorded which sample is in each position in the PCR machine.

Reaction Conditions:

1 cycle:

       94oC, 3 minutes

5 cycles:

       94oC, 30 seconds
       69oC, 30 seconds
       72oC, 1 minute

5 cycles:

       94oC, 30 seconds
       65oC, 30 seconds
       72oC, 1 minute

20 cycles:

       94oC, 30 seconds
       61oC, 30 seconds
       72oC, 1 minute

1 cycle:

       72oC, 3 minutes

While the reactions are being performed, you can proceed to setting up the Finish PCR reactions.

Finish PCR

Templateless PCR produces a relatively low yield of the full-length gene; many smaller fragments that did not complete assembly are also produced. The goal of PCR is to exponentially amplify a template DNA sequence to increase its abundance (A good introduction to PCR is here: http://www.dnalc.org/resources/animations/pcr.html). The goal of finish PCR is to exponentially amplify the full-length gene that was assembled by templateless PCR. The result is that most of the DNA present in the reaction mixture following finish PCR will be the full-length gene.

• Set up your reactions on ice. Keep nucleotides and master mix on ice AT ALL TIMES!
• Be sure that each reagent is well mixed by pipetting up and down several times before using

You will be performing 3 reactions:
1. Amplification of the GFP gene
2. A positive http://openwetware.org/skins/common/images/button_bold.pngcontrol reaction (a reaction that we know should produce large amounts of DNA)
3. A negative control reaction (a reaction that contains no input DNA and therefore should not produce any DNA; this is a check for contaminating DNA)

Procedure:
1. Obtain 3 PCR tubes (these are the small thin-walled tubes). Label the tubes 1-3.
2. To each tube, add the components listed below. You will need to wait until the templateless PCR reactions have completed to add the GFP template.

	Amplification of GFP gene	Positive control reaction	Negative control reaction
PCR master mix (buffer and enzyme)	10 ul	10 ul	10 ul
Nucleotides (1.25 mM)	5 ul	5 ul	5 ul
Oligos	5 ul of amplification oligos	5 ul of control oligos	5 ul of control oligos
DNA template	5 ul of GFP template	5 ul of PC template	5 ul of water
Total	25 ul	25 ul	25 ul

3. Once the templateless PCR reactions have finished, obtain a 1.7 ml microcentrifuge tube. Label tube with you initials and “T-PCR NC”. Transfer the liquid from tube 3 of your templateless PCR (the negative control) to this tube and put it away to be used in two weeks.
4. Obtain a 1.7 ml microcentrifuge tube. Label tube with your initials and “T-PCR PC”. Transfer the liquid from tube 2 of your templateless PCR (the positive control) to this tube and put it away to be used in two weeks.
5. Obtain a 1.7 ml microcentrifuge tube. Label tube with your initials and “GFP template”. Transfer the liquid from tube 1 of your templateless PCR (the assembly of the GFP gene) to this tube. To this tube, add 180 ul of water. Mix well and use 5 ul of this mixture as the DNA template for Finish PCR reaction 1 according to the table above.
6. Cap your tubes and make sure that they are sealed tightly so that the liquid will not evaporate.
7. Place your tubes in the PCR machine in the position for which you signed up. Make sure that you have recorded which sample is in each position in the PCR machine.

Reaction Conditions:

1 cycle:

      94oC, 3 minutes

35 cycles:

       94oC, 30 seconds
       55oC, 30 seconds
       72oC, 1 minute

1 cycle:

       72oC, 3 minutes