Haynes:Making BioBricks
Making Standardized DNA Parts
- Double-stranded Oligo Insert: A part that is smaller than ~85 bp can be made into an oligonucleotide insert.
- Overlapping Oligos: A part that is between ~ 85 - 150 bp can be assembled from smaller overlapping oligonucleotides.
- PCR Amplification: For a part that is larger than ~150 bp and is based on an existing DNA fragment, use PCR amplification of the existing DNA.
Double-stranded Oligo Insert
1. Design your oligos: An oligo insert should have a XbaI sticky end upstream of the part, SpeI and NotI sites downstream, and a PstI sticky end downstream.
| Sense oligo: | 5' CTAGA[coding sequence]ACTAGTAGCGGCCGCTGCA 3' |
| Anti-sense oligo: | 5' GCGGCCGCTACTAGT[reverse complement of coding sequence]T 3' |
Double stranded result:
5' ctaga [coding sequence] actagt a gcggccgctgca 3'
| ||||||||||||||||| |||||| | ||||||||
3' t [rev. comp. seq.] tgatca t cgccggcg 5'
2. Have the oligos synthesized (you can order through a company like www.idtdna.com).
Troubleshooting note: order the oligos with 5’ phosphates (optional) if you are having difficulty cloning.
3. Set up an annealing reaction as follows:
| Sense oligo 1 (100 μM) | 3.0 μl |
| Anti-sense oligo (100 μM) | 3.0 μl |
| 10x annealing buffer* | 2.0 μl |
| dH2O | 12.0 μl |
| nbsp; | 50 μl |
Heat at 100°C for 5 min., remove the entire heat block or water bath from the heat source, and allow to cool slowly to room temperature.
--> *10x annealing buffer: 1 M NaCl; 100 mM Tris-HCl, pH 7.4
4. If you need to calculate the amount of insert needed to set up a specific ratio of insert to vector for the ligation, use this formula to estimate ng/μl of the oligo insert:
[(total ng stock oligo 1 / μl dH2O used to dissolve dry oligo 1 + total ng stock oligo 2 /
μl dH2O used to dissolve dry oligo 2) * 3 μl] / 20 μl
5. Ligate the double-stranded insert into a linearized vector with XbaI and PstI ends and transform into E. coli. (use any standard ligation/ transformation protocol)
Overlapping Oligos
1. Design your oligos: You can use software, such as “Oligo Cuts” (L. Harden, Davidson College, http://gcat.davidson.edu/IGEM06/oligo.html), to determine the optimal length and number of oligos for building your BioBrick. An example is shown below:
Ligating a “front insert” in a “front vector” to make A+B
Ligating a “back insert” in a “back vector” to make the same A+B construct
Note: After DNA plasmids are cut, the desired DNA fragments are isolated and purified using agarose gel electrophoresis (ref). The fragments that are bordered by a dotted line are discarded.
A few important words about DNA assembly: In the diagram above, all of the DNA starts out as a plasmid (circular piece of DNA). Each plasmid has a region called a backbone (shown as a thin arc) that consists of the origin of replication (the DNA sequence that encodes information for making copies of the plasmid; the mechanism is very complex) and an antibiotic resistance gene (e.g., ampicillin resistance). After the plasmids are cut with restriction enzymes (ref), one DNA fragment is used as an insert (no backbone) and another is used as the vector (has the backbone). The final DNA ligation product (the recombinant plasmid) must have one backbone.
About the "S/X" scar sequence: When two BioBricks are ligated, the SpeI end ligates with the XbaI end. The resulting sequence is a mixed site (called a scar) that cannot be re-cut by any enzyme. Silver Standard assembly produces a 6 b.p. scar (two codons) so that proteins can be fused together without creating a frame-shift mutation.
