Materials
- Oligos
- Buffer
- T4 PNK
- Water
Double-Stranded Oligo Insert
- 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.
- Have the oligos synthesized (you can order through a company like www.idtdna.com). They do not require 5' phosphates.
- Set up an annealing/ phosphorylation reaction as follows with the following cycling parameters:
Thermal cycler program:
-37°C, 30 min
-95°C, 5 min
-Ramp down to 25°C, 5°C/1 min. [90/1 min, 85/1 min, 80/1 min, ... 25°C/1 min]
-25°C, ∞
| Sense oligo (100 μM) | 1.0 μl |
| Anti-sense oligo (100 μM) | 1.0 μl |
| 10x T4 ligation Buffer (NEB) | 1.0 μl |
| T4 PNK (NEB) | 0.5 μl |
| dH2O | 6.5 μl |
| 10 μl |
- Dilute the product(s) 1:250. (Add 2 μL product to 498 μL dH2O)
- 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)
- Note: Using 2.0 μL of the 1:250 diluted dsOligo per 50 - 100ng vector works well in our hands. If you prefer 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) * 1.0 μl] / 20 μl]
Overlapping Oligos
- Design your oligos: You can use software, such as “The Oligator” (Davidson College, http://gcat.davidson.edu/igem10/), to determine the optimal length and number of oligos for building your BioBrick. An example of Oligator output is shown below:
- 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.
- 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: 1M NaCl; 100mM Tris-HCl, pH 7.4
- 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]
- 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)
PCR Amplification
- Design your primers - XbaI/PstI cloning: (or see alternative below) The following primers will generate a XbaI site upstream of your part, and SpeI, NotI, and PstI sites downstream of your part (with extra bases at each end to aid restriction digestion). Since the next step will involved XbaI/PstI cloning, EcoRI and NotI are omitted to avoid wasting DNA (nd synthesis costs)
| Forward primer: | 5' | ccttTCTAGA | [15-20 bp of the coding strand] | 3' |
| Reverse primer: | 5' | aaggCTGCAGCGGCCGCtACTAGT | [15-20 bp reverse complement] | 3' |
- Design your primers - EcoRI/SpeI cloning: Alternatively, you can generate a PCR product with EcoRI, NotI, and XbaI upstream and SpeI downstream. You may want to use this approach if your BioBrick happens to contain an illegal (e.g. PstI) site, which often happens for eukaryotic protein coding regions.
| Forward primer: | 5' | ccttGAATTCGCGGCCGCaTCTAGA | [15-20 bp of the coding strand] | 3' |
| Reverse primer: | 5' | aaggACTAGT | [15-20 bp reverse complement] | 3' |
- Have the primers (oligos) synthesized. You can order through a company like www.idtdna.com.
- Follow the PCR Clip and Clone protocol to clone the PCR fragment into a BioBrick vector in RFC23 format (e.g. V0120).