One-Step Gene Assembly
(Reference: Gang Wu, Julie B. Wolf, Ameer F. Ibrahim, Stephanie Vadasz, Muditha Gunasinghe and Stephen J. Freeland, Simplified gene synthesis: A one-step approach to PCR-based gene construction, Journal of Biotechnology, 124(3):496-503)
- Design overlapping oligonucleotides, generally 40 bases in length, that encompass the sense strand of the gene of interest. Design antisense oligonucleotides that stagger the sense oligos by 20 bases.
- Design outside PCR amplification primers to incorporate the appropriate restriction enzyme recognition sites, if desired, and to overlap the assembled gene sequence by at least 15 nucleotides. (Order oligonucleotides from IDT or Invitrogen using their standard desalting purification).
- Reconstitute oligonucleotides to 100 µM in 10 mM Tris pH=8.5 (same as Qiagen buffer EB). Vortex well to reconstitute and store at -20°C.
- Prepare a gene assembly mix by combining 5 µl of each of the gene assembly oligos. Dilute this mix so that each oligo is at a final concentration of 1 µM. (This will be referred to as 1X). Then dilute this mix 1:2 (0.5X), 1:5 (0.2X) and 1:10 (0.1X) in Tris buffer. This step is to optimize the assembly/amplification of the required product which varies from one gene to the next – so best to set up 4 different reactions, one for each dilution of the mix, so determine which gives you the best yield and the least background.
- Prepare 10 µM dilutions of outside amplification primers.
- Perform one step gene assembly/amplifications using the following:
Reaction conditions:
Template |
5 µl gene assembly mix (1X, 0.5X, 0.2X, and 0.1X) |
Primers (0.4 uM final) |
2 µl of 10 µM outside amplification primer #1 (OP-5’)
2 µl of 10 µM outside amplification primer #2 (OP-3’) |
dNTP (0.2 mM final) |
5 µl of 2 mM each (provided with KOD enzymes) |
10X PCR buffer |
5 µl (provided with KOD enzymes) |
25 mM MgCl2 |
2 µl for KOD HiFi; none needed for XL |
Sterile water |
28 µl for KOD HiFi; 30 µl for XL |
KOD HiFi enzyme for highest accuracy or KOD XL for TA cloning |
0.4 µl KOD HiFi (Novagen) or
1 µl for KOD XL (Novagen) |
Total |
50 µl |
Cycling conditions:
|
KOD XL– up to 2 kb |
KOD HiFi – up to 2 kb |
25 cycles |
94°C |
30 sec |
98°C |
15 sec |
|
52°C |
5 sec |
52°C |
2 sec |
|
72°C |
30-60 sec/kb |
72°C |
20 sec |
1 cycle |
74°C |
10 min |
|
|
- Analyze 5µl on agarose gel.
- Purify remainder using Qiagen PCR purification columns, digest overnight, then gel purify. Ligate to vector of choice.
See Sample Results
This Web page is maintained by Julie B. Wolf, UMBC;
Last updated on
3/2/2010
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