Gene Synthesis from Oligonucleotides: Difference between revisions
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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: [http://en.wikipedia.org/wiki/Polymerase_cycling_assembly] | 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: [http://en.wikipedia.org/wiki/Polymerase_cycling_assembly] | ||
<br> | |||
*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<br> | |||
<br> | |||
'''You will be performing 3 reactions:''' | |||
#Assembly of the GFP gene | |||
#A positive control reaction (a reaction that we know should produce large amounts of DNA) | |||
#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) | |||
<br> | |||
'''Procedure:''' | |||
1. Obtain 3 PCR tubes (these are the small thin-walled tubes). Label the tubes 1-3.<br> | |||
2. To each tube, add the components listed below.<br> | |||
<br> | |||
{| border="1" | |||
|- | |||
| || 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 | |||
|} | |||
<br> | |||
3. Cap your tubes and make sure that they are sealed tightly so that the liquid will not evaporate.<br> | |||
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.<br> | |||
<br> | |||
'''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. |
Revision as of 18:13, 6 July 2014
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:
- Assembly of the GFP gene
- A positive control reaction (a reaction that we know should produce large amounts of DNA)
- 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.