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'
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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
[ Oligo A ][ other ][ other ][ Oligo B ]
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[ Oligo D ][ other ][ Oligo C ]
Add the following modifications to the oligos that were generated by the program:
| 5’ sense oligo (A): | 5’ CTAGA [oligo sequence] 3’ |
| 3’ sense oligo (B): | 5’ [oligo sequence] ACTAGTAGCGGCCGCTGCA 3’ |
| 5’ anti-sense oligo (C): | 5’ GCGGCCGCTACTAGT [oligo sequence] 3’ |
| 3’ anti-sense oligo (D): | 5’ [oligo sequence] T 3’ |
The final insert will have a XbaI sticky end upstream of the part, SpeI and NotI sites downstream, and a PstI sticky end downstream.
2. Have the oligos synthesized (you can order through a company like www.idtdna.com). Note: To avoid self-ligation of the oligo insert during the final ligation, do not add 5’ phosphates to the oligos. DNA nicks in the insert-vector ligation will be repaired via plasmid replication in E. coli.
3. Set up an annealing reaction as follows:
| Oligo (100 μM) | 3.0 μl of each |
| 10x annealing buffer* | 2.0 μl |
| dH2O | ____ μl |
| 20 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. 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 + …n) * 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)
